Use of a BMP protein receptor complex for screening bone metabolism actives and cells co-transfected with a type II BMP receptor and a type I BMP receptor

ABSTRACT

The present invention relates to a method for determining whether a compound is capable of binding to a BMP receptor kinase protein complex, the method comprising introducing a sample comprising the compound to the BMP receptor kinase protein complex and allowing the compound to bind to the BMP receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and the BMP receptor kinase protein BRK-3. The invention further relates to a method for determining the concentration of a BMP receptor ligand in a clinical sample, the met-hod comprising introducing the sample comprising the ligand to a BMP receptor kinase protein complex and allowing the ligand to bind to the BMP receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and BMP receptor kinase protein BRK-3. The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the BMP receptor kinase protein BRK-3 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein. The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for a soluble or incomplete BMP type I receptor kinase protein and a soluble or incomplete BMP receptor kinsase protein BRK-3. The invention further relates to a method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex.

This application is a divisional of U.S. patent application Ser. No.08/462,467, filed Jun. 5, 1995, now Issued as U.S. Pat. No. 6,210,899issued on Apr. 3, 2001, and which application is a CIP of Ser. No.08/334,178 filed Nov. 4, 1994, now abandoned.)

TECHNICAL FIELD

The present invention relates to the field of bone formation anddevelopment. Specifically, the present invention relates to the use of anew bone morphogenetic protein type II receptor, together with a bonemorphogenetic type I receptor, for screening bone metabolism actives.The invention further relates to cells co-transfected with DNA codingfor this receptor and DNA coding for a type I bone morphogenetic proteinreceptor.

BACKGROUND

Humans and other warm-blooded animals can be afflicted by a number ofbone-related disorders. Such disorders range from bone fractures, todebilitating diseases such as osteoporosis. While in healthy individualsbone growth generally proceeds normally and fractures heal without theneed for pharmacological intervention, in certain instances bones maybecome weakened or may fail to heal properly. For example, healing mayproceed slowly in the elderly and in patients undergoing treatment withcorticosteroids (e.g., transplant patients). Osteoporosis is a conditionin which bone hard tissue is lost disproportionately to the developmentof new hard tissue. Osteoporosis can generally be defined as thereduction in the quantity of bone, or the atrophy of skeletal tissue;marrow and bone spaces become larger, fibrous binding decreases, andcompact bone becomes fragile. Another bone related disorder isosteoarthritis, which is a disorder of the movable joints characterizedby deterioration and abrasion of articular cartilage, as well as byformation of new bone at the joint surface.

While a variety of treatments are available for such bone-relateddisorders, none of the treatments provide optimum results. One of thedifficulties facing individuals who treat bone-related disorders is alack of complete understanding of bone metabolism and of thebone-related disorders. A key to such understanding is identifying andcharacterizing each of the components involved in bone growth. Bonemorphogenetic proteins (BMPs) have been demonstrated to play a role inbone formation and development (J. M. Wozney, Molec. Reproduct. andDevelop., 32: 160-167 (1992)).

Furthermore, the role of BMPs may not be limited to their role in bone.The finding that the BMPs are found at significant concentrations inother tissues such as brain, kidney, stratified squamous epithelia, andhair follicle (N. A. Wall, M. Blessing, C. V. E. Wright, and B. L. M.Hogan, J. Cell Biol., 120: 493-502 (1993); E. Özkaynak, P. N. J.Schnegelsberg, D. F. Jin, G. M. Clifford, F. D. Warren, E. A. Drier, andH. Oppermann, J. Biol. Chem., 267: 25220-25227 (1992); K. M. Lyons, C.M. Jones, and B. L. M. Hogan, Trends in Genetics, 7: 408-412 (1991); V.Drozdoff, N. A. Wall, and W. J. Pledger, Proceedings of the National.Academy of Sciences, U.S.A., 91: 5528-5532 (1994)) suggests that theymay play additional roles in development and differentiation. In supportof this, BMPs have recently been found to promote nerve celldifferentiation and to affect hair follicle formation (K. Basler, T.Edlund, T. M. Jessell, and T. Yamada, Cell, 73: 687-702 (1993); V. M.Paralkar, B. S. Weeks, Y. M. Yu, H. K. Kleinman, and A. H. Reddi, J.Cell Biol., 119: 1721-1728 (1992); M. Blessing, L. B. Nanney, L. E.King, C. M. Jones, and B. L. Hogan, Genes Dev., 7: 204-215 (1993)).

A BMP initiates its biological effect on cells by binding to a specificBMP receptor expressed on the plasma membrane of a BMP-responsive cell.A receptor is a protein, usually spanning the cell membrane, which bindsto a ligand from outside the cell, and as a result of that binding sendsa signal to the inside of the cell which alters cellular function. Inthis case, the ligand is the protein BMP, and the signal induces thecellular differentiation.

Because of the ability of a BMP receptor to specifically bind BMPs,purified BMP receptor compositions are useful in diagnostic assays forBMPs, as well as in raising antibodies to the BMP receptor for use indiagnosis and therapy. In addition, purified BMP receptor compositionsmay be used directly in therapy to bind or scavenge BMPs, therebyproviding a means for regulating the activities of BMPs in bone andother tissues. In order to study the structural and biologicalcharacteristics of BMP receptors and the role played by BMPs in theresponses of various cell populations to BMPs during tissuegrowth/formation stimulation, or to use a BMP receptor effectively intherapy, diagnosis, or assay, purified compositions of BMP receptor areneeded. Such compositions, however, are obtainable in practical yieldsonly by cloning and expressing genes encoding the receptors usingrecombinant DNA technology. Efforts to purify BMP receptors for use inbiochemical analysis or to clone and express mammalian genes encodingBMP receptors have been impeded by lack of a suitable source of receptorprotein or mRNA. Prior to the present invention, few cell lines wereknown to express high levels of high affinity BMP receptors whichprecluded purification of the receptor for protein sequencing orconstruction of genetic libraries for direct expression cloning.Availability of the BMP receptor sequence will make it possible togenerate cell lines with high levels of recombinant BMP receptor forbiochemical analysis and use in screening experiments.

The BMPs are members of the TGF-β superfamily. Other members of theTGF-β superfamily include TGF-β, activins, inhibins, MüllerianInhibiting Substance, and the Growth and Differentiation Factors (GDFs).As expected, the receptors for various members of the TGF-β superfamilyshare similar structural features. Receptors of the TGF-β ligandsuperfamily are typically classified into one of two sub-groups,designated as type I and type II. The type I and type II receptors areclassified as such based on amino acid sequence characteristics. Boththe type I and type II receptors possess a relatively smallextracellular ligand binding domain, a transmembrane region, and anintracellular protein kinase domain that is predicted to haveserine/threonine kinase activity (Lin and Moustakas, Cellular andMolecular Biology, 40: 337-349 (1994); L. S. Mathews, Endocrine Reviews,15: 310-325 (1994); L. Attisano, J. L. Wrana, F. López-Casillas, and J.Massagué, Biochimica et Biophysica Acta, 1222: 71-80 (1994)).

The type I receptors cloned to date belong to a distinct family whosekinase domains are highly related and share >85% sequence similarity (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)).The intracellular juxtamembrane region of the type I receptors ischaracterized by an SGSGSG motif 35-40 amino acids from thetransmembrane region, and the carboxy terminus of these receptors isextremely short (B. B. Koenig et al., Molecular and Cellular Biology,14: 5961-5974 (1994); L. Attisano, J. L. Wrana, F. López-Casillas, andJ. Massagué, Biochimica et Biophysica Acta, 1222: 71-80 (1994)). Theextracellular domain of the type I receptors contains a characteristiccluster of cysteine residues, termed the “cysteine box”, located within25-30 amino acids of the transmembrane region, and another cluster ofcysteine residues, termed the “upstream cysteine box”, located after theputative signal sequence (B. B. Koenig, et al., Molecular and CellularBiology, 14: 5961-5974 (1994); L. Attisano, et al., Biochimica etBiophysica Acta, 1222: 71-80 (1994)).

In contrast to the type I receptors, the kinase domains of the type IIreceptors are only distantly related to one another. The SGSGSG motiffound in type I receptors is not found in type II receptors. Also, the“upstream cysteine box” of type I receptors is not present in type IIreceptors. Furthermore, while all of the activin type II receptorscontain a proline-rich sequence motif in the intracellular juxtamembraneregion, there is no characteristic sequence motif that is common to alltype II receptors (L. S. Mathews, Endocrine Reviews, 15: 310-325(1994)). The length of the carboxy terminus of the type II receptors isconsiderably variable, with the longest known carboxy terminus beingfound in the BMP type II receptor, DAF-4 (M. Estevez, L. Attisano, J. L.Wrana, P. S. Albert, J. Massagué, and D. L. Riddle, Nature, 365: 644-49(1993)), that was cloned from the nematode C. elegans. The extracellulardomain of the type II receptors contains a single cysteine box locatednear the transmembrane region. Aside from the presence of the cysteinebox, there is little sequence similarity amongst the extracellulardomains of the type II receptors for TGF-β, activin, and BMPs.

Signaling by members of the TGF-β ligand superfamily requires thepresence of both type I and type II receptors on the surface of the samecell (L. S. Mathews, Endocrine Reviews, 15: 310-325 (1994); L. Attisano,J. L. Wrana, F. López-Casillas, and J. Massagué, Biochimica etBiophysica Acta, 1222: 71-80 (1994)). The BMPs are members of the TGF-βligand superfamily; given the high degree of structural similarity amongthese family members, it is expected that their receptors will bestructurally and functionally related to the TGF-β and activinreceptors. It is anticipated that, like the TGF-β and activin receptorsystems (J. Massagué, L. Attisano, and J. L. Wrana, Trends in CellBiology, 4: 172-178 (1994)), both a BMP type I receptor and a BMP typeII receptor will be needed in order to transduce a BMP signal within acell or tissue. Hence, there is a need for a mammalian type II BMPreceptor kinase protein in addition to the type I receptors that havealready been cloned.

Three distinct mammalian type I receptors have been reported for theBMPs: Bone Morphogenetic Protein Receptor Kinase-1 (herein referred toas “BRK-1”) (see U.S. Ser. No. 08/158,735, filed Nov. 24, 1993 by J. S.Cook, et al.; and B. B. Koenig et al., Molecular and Cellular Biology,14: 5961-5974 (1994)), ALK-2, and ALK-6. BRK-1 is the mouse homologue ofALK-3, which has also been demonstrated to bind BMP-4, as does ALK-6;ALK-2 binds BMP-7 (see P. ten Dijke, H. Yamashita, T. K. Sampath, A. H.Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C. -H. Heldin, and K.Miyazono, J. Biological Chemistry, 269: 16985-16988 (1994)). It is alsopostulated that ALK-6 is the mouse homologue of the chicken receptorBone Morphogenetic Protein Receptor Kinase-2 (herein referred to as“BRK-2”) (also referred to as RPK-1) (S. Sumitomo, T. Saito, and T.Nohno, DNA Sequence, 3: 297-302 (1993)). The rat homologue of BRK-1 hasalso been cloned, as BMPR-Ia (K. Takeda, S. Oida, H. Ichijo, T. Iimura,Y. Marnoka, T. Amagasa and S. Sasaki, Biochemical and BiophysicalResearch Communications, 204: 203-209 (1994)).

In co-pending application U.S. Ser. No. 08/334,179, filed Nov. 4, 1994by Rosenbaum and Nohno, a novel mammalian BMP type II receptor (referredto as “BRK-3”) is described and claimed. Prior to the cloning of theBRK-3 receptor, the only type II receptor for BMP-2 and BMP-4, namedDAF-4, was cloned from the nematode C. elegans (M. Estevez, L. Attisano,J. L. Wrana, P. S. Albert, J. Massagué, and D. L. Riddle, Nature, 365:644-9 (1993)). Because of the large evolutionary distance between thenematode and mammals, it has not been possible to use the DAF-4 cDNA asa probe with which to clone the mammalian DAF-4 homologue. This impliesthat the DNA sequence of the mammalian type II receptor for BMPs issubstantially divergent from that of DAF-4, and it was thereforenecessary to clone a mammalian type II receptor for the BMPs.

The BMP receptor kinase protein BRK-3 of the co-pending applicationprovides a mammalian type II receptor which enables the formation of acomplex with a BMP type I receptor. This complex, which is described indetail below, is capable of binding BMPs with high affinity, and istherefore useful for identifying compounds having BMP receptor affinity.The complex of the present invention will also enable the formation of ahigh affinity complex that is competent for signaling a response to BMPsin concert with the mammalian type I receptor(s) for BMPs. The mammalianBMP receptor complex is therefore more relevant for the identificationof novel compounds which interact with the BMP receptor, and which willbe useful as therapeutic agents in humans and other mammals, than is areceptor complex that is composed of the nematode type II receptor andthe mammalian type I receptor.

OBJECTS OF THE PRESENT INVENION

It is an object of the present invention to provide a method foridentifying compounds capable of binding to a BMP receptor kinaseprotein complex.

It is also an object of the present invention to provide a method fordetermining the amount of a compound capable of binding a BMP receptorkinase protein complex in a sample.

It is also an object of the present invention to provide a host cellcomprising a recombinant expression vector encoding a BMP type IIreceptor kinase protein and a recombinant expression vector encoding aBMP type I receptor kinase protein comprising said BMP receptor kinaseprotein complex.

It is also an object of the present invention to provide a method fordetermining whether a test compound produces a signal upon binding to aBMP receptor protein complex.

SUMMARY

The present invention relates to a method for determining whether acompound is capable of binding to a BMP receptor kinase protein complex,the method comprising introducing a sample comprising the compound tothe BMP receptor kinase protein complex and allowing the compound tobind to the BMP receptor kinase protein complex, wherein the BMPreceptor kinase protein complex is comprised of a BMP type I receptorkinase protein and the BMP receptor kinase protein BRK-3.

The invention further relates to a method for determining theconcentration of a BMP receptor ligand in a clinical sample, the methodcomprising introducing the sample comprising the ligand to a BMPreceptor kinase protein complex and allowing the ligand to bind to theBMP receptor kinase protein complex, wherein the BMP receptor kinaseprotein complex is comprised of a BMP type I receptor kinase protein andBMP receptor kinase protein BRK-3.

The invention further relates to a host cell co-transfected with anexpression vector comprising a DNA sequence that codes for the BMPreceptor kinase protein BRK-3 and an expression vector comprising a DNAsequence that codes for a BMP type I receptor kinase protein.

The invention further relates to a host cell co-transfected with anexpression vector comprising a DNA sequence that codes for a soluble BMPtype I receptor kinase protein and a soluble BMP receptor kinsaseprotein BRK-3.

The invention further relates to a host cell co-transfected with anexpression vector comprising a DNA sequence that codes for an incompleteBMP type I receptor kinase protein and an incomplete BMP receptorkinsase protein BRK-3.

The invention further relates to a method for determining a testcompound produces a signal upon binding to a BMP receptor proteincomplex, the method comprising: (a) labeling BMP receptor proteincomplex expressing cells with ³²P, wherein the cells have beentransfected with a DNA sequence coding for BMP receptor kinase proteinBRK-3 and a DNA sequence coding for a BMP type I receptor kinaseprotein; (b) culturing (i) a first set of the cells in the presence ofthe test compound, and (ii) a second set of the cells in the absence ofthe test compound; (c) quantitating via autoradiography anyphosphorylated proteins produced from step (b); and (d) comparing theamount of phosphorylated proteins quantitated in step (c) from the firstset of cells to the amount of phosphorylated proteins quantitated instep (c) for the second set of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence of the degenerate oligonucleotide primersused in the PCR amplification of t-BRK-3. The nucleotide bases adenine,thymine, cytosine, and guanine are represented by A, T, C and Grespectively. The letter N represents the presence of an equal mixtureof A, T, C, and G at that site. The primers are derived from thesequence of the TGF-β type II receptor (H. Y. Lin, X. F. Wang, E.Ng-Eaton, R. A. Weinberg, and H. F. Lodish, Cell, 68: 775-785 (1992)).

FIG. 2 shows the construct pJT4-hBRK3T, used for transient mammalianexpression of t-BRK-3. CMV, cytomegalovirus early promoter/enhancer; R,the “R” element from the long terminal repeat of human T-cell leukemiavirus-1; SP, an intron splice site from the SV40 virus; T3, promoterregion from the T3 bacteriophage; T7, promoter region from the T7bacteriophage; poly A, region from the SV40 virus directingpolyadenylation of the message; SV40 ORI, origin of replication from theSV40 virus; Amp, ampicillin resistance gene for selection in E. coli.

FIG. 3 shows the construct pJT4-J159F, used for transient mammalianexpression of BRK-1. Abbreviations are the same as those in FIG. 2.

FIG. 4 shows the construct pJT3-BRK2, used for transient mammalianexpression of BRK2. Abbreviations are the same as those in FIG. 2.

FIG. 5 shows the construct pJT4-Daf4, used for transient mammalianexpression of the C. elegans receptor DAF-4. Abbreviations are the sameas those in FIG. 2.

FIG. 6 shows whole cell binding of [¹²⁵I]-BMP-4 to t-BRK-3 expressed inCOS-7 cells, in the presence or absence of the type I receptors BRK-1and BRK2. Bars represent specific binding of [¹²⁵I]-BMP-4, normalized tocell number. Left to right, NIH3T3 embryonic fibroblasts; COS-7 cells;COS-7 cells transfected with the vector pJT-4 alone (designated “mock”);COS-7 cells transfected with BRK-1 alone, BRK-1 plus 10 or 20 μg oft-BRK-3, BRK-2 alone, BRK-2 plus 10 or 20 μg of t-BRK-3, and t-BRK-3alone (20 μg).

FIG. 7 shows crosslinking of [¹²⁵I]-BMP-4 to COS-1 cells transfectedwith t-BRK-3, in the presence or absence of the type I receptors BRK-1and BRK-2. Molecular weight standards are shown on the left. Labels onthe right indicate the bands which migrate at the predicted molecularweights of t-BRK-3, BRK-1, and BRK-2 crosslinked to [¹²⁵I]-BMP-4. Leftto right, the lanes represent COS-1 cells transfected with BRK-1 alone;BRK-1 plus 2 μg/ml t-BRK-3; BRK-1 plus 4 μg/ml t-BRK-3; BRK-2 alone;BRK-2 plus 2 μg/ml t-BRK-3; BRK-2 plus 4 μg/ml t-BRK-3; t-BRK-3 alone at2 μg/ml; and t-BRK alone at 4 μg/ml. Volume of DNA mixture is 4 ml. Inthis figure, “BRK-3*” is t-BRK-3.

FIG. 8 shows an immunoprecipitation of t-BRK-3 and the C. elegans typeII receptor DAF-4 expressed in COS-1 cells and crosslinked to[¹²⁵I]-BMP-4 in the presence or absence of the type I receptors BRK-1 orBRK-2. Molecular weight standards are shown on the left; areas shown atthe right indicate labeled protein bands migrating at the predictedmolecular weight of DAF4, t-BRK-3, BRK-1, or BRK-2 crosslinked to[¹²⁵I]-BMP-4. Antiserum 1379 was used for COS-1 cells transfected withBRK-1 in the presence or absence of type II receptors, and antiserum1380 for COS-1 cells transfected with BRK-2 in the presence or absenceof type II receptors. For all others, antiserum is listed inparentheses. Left to right, NIH3T3 embryonic fibroblasts (1379),followed by COS-1 cells transfected with BRK-1 alone; BRK-1 plus DAF4;BRK-1 plus t-BRK-3; BRK-2 alone; BRK-2 plus DAF-4; BRK-2 plus t-BRK-3.This is followed by NIH3T3 cells (1380), followed by COS-1 cellstransfected with DAF4 alone (1379), and t-BRK-3 alone (1380). In thisfigure, “BRK-3*” is t-BRK-3.

FIG. 9 shows an immunoprecipitation of COS-1 cells transfected withBRK-2 and t-BRK-3 and crosslinked to [¹²⁵I]-BMP-4 at a concentration of210 pM, in the presence or absence of excess unlabeled competitors asindicated. Antiserum 1380 is used. Duplicate lanes at left show nounlabeled competitor added, followed by addition of (left to right) 10nM BMP-4; 10 nM BMP-2; 10 nM DR-BRMP-2; and 50 nM TGF-β₁. In thisfigure, “BRK-3*” is t-BRK-3.

FIG. 10 shows the construct pJT6-mBRK-3L, used for transient mammalianexpression of mouse BRK-3. Abbreviations used are the same as those forFIG. 2.

FIG. 11 shows the construct pJT6-mBRK-3 S, used for transient mammalianexpression of mouse BRK-3. In this construct, most of the untranslated3′ region has been removed. Abbreviations used are the same as those forFIG. 2.

FIG. 12 shows whole cell binding of [¹²⁵I]-BMP-4 to mouse BRK-3expressed in COS-1 cells, in the presence or absence of the type Ireceptor BRK-2. Bars represent specific binding of [¹²⁵I]-BMP-4,normalized to cell number. Constructs used for mouse BRK-3 arepJT6-mBRK-3L and pJT6-mBRK-3S; for BRK-2, the construct is pJT3-BRK-2.Both constructs contain the complete coding region of mouse BRK-3. InpJT6-mBRK-3 S, an A-T rich region in the 3′ untranslated region has beendeleted. Left to right, COS-1 cells transfected with the vector pJT-6alone (designated “mock”); pJT3-BRK-2 alone; the construct pJT6-mBRK-3Salone; pJT6-mBRK-3L alone; pJT3-BRK-2 plus pJT6-BRK-3S; and pJT3-BRK-2plus pJT6-BRK-3L.

FIG. 13 shows crosslinking of [¹²⁵I]-BMP-4 to m-BRK-3 in the presenceand absence of type I BMP receptors. COS-1 cells are transfected withthe cDNA for BRK-3 using the construct pJT6-mBRK-3S, and/or with cDNAsfor BRK-1 (using pJT4-J159F) or BRK-2 (using pJT3-BRK-2). The cells arethen allowed to bind [¹²⁵I]-BMP-4, crosslinked with disuccinimidylsuberate, and subjected to SDS gel electrophoresis. Position ofmolecular weight standards is indicated on the left. Left to right:COS-1 cells transfected with BRK-1 alone; BRK-1 plus m-BRK-3; m-BRK-3alone; BRK-2 plus m-BRK-3; BRK-2 alone; and vector alone. Bandsidentified with BRK-1, BRK-2, and BRK-3 are indicated on the right.

FIG. 14 shows immunoprecipitation of m-BRK-3 in the presence and absenceof type I BMP receptors. COS-1 cells are transfected with the cDNA form-BRK-3 using the construct pJT6-mBRK-3S, and/or with cDNAs for BRK-1(using pJT4-J159F) or BRK-2 (using pJT3-BRK-2). The cells are thenallowed to bind [¹²⁵I]-BMP-4, crosslinked with disuccinimidyl suberate,immunoprecipitated with antibodies to BRK-1 or BRK-2, and subjected toSDS gel electrophoresis. Antisera used are indicated below the lanes:PI, preimmune; 1379, for cells transfected with cDNA for BRK-1; 1380,for cells transfected with cDNA for BRK-2. Position of molecular weightstandards is indicated on the left. Left to right, COS-1 cellstransfected with BRK-1 plus m-BRK-3 (preimmune serum); BRK-1 alone;BRK-1 plus m-BRK-3; BRK-2 plus m-BRK-3; BRK-2 alone; and BRK-2 plusm-BRK-3 (preimmune serum).

FIG. 15 shows a map of the insert of pHSK1040. This construct containsthe complete coding region of human BRK-3 in BLUESCRIPT II SK (−).

DESCRIPTION

The present invention answers the need for a method for determiningwhether a compound has BMP receptor affinity. The method comprisesintroducing a sample comprising a test compound to a BMP receptor kinaseprotein complex and allowing the compound to bind to the BMP receptorkinase protein complex, wherein the receptor complex comprises a BMPtype I receptor kinase protein and the BMP type II receptor kinaseprotein designated herein as “BRK-3”. The invention also answers theneed for a host cell that is co-transfected with an expression vectorcomprising a DNA sequence that codes for BMP receptor kinase proteinBRK-3 and an expression vector comprising a DNA sequence that codes fora BMP type I receptor kinase protein. Also provided is a method fordetermining the concentration of a BMP receptor ligand in a clinicalsample, the method comprising introducing the sample comprising theligand to a BMP receptor kinase protein complex and allowing the ligandto bind to the receptor complex, wherein the receptor complex iscomprised of a BMP type I receptor kinase protein and BMP receptorkinase protein BRK-3. The invention also answers the need for a hostcell that is co-transfected with an expression vector comprising a DNAsequence that codes for a soluble BMP receptor kinase proten BRK-3 andan expression vector comprising a DNA sequence that codes for a solubleBMP type I receptor kinase protein. The invention also answers the needfor a host cell that is co-transfected with an expression vectorcomprising a DNA sequence that codes for an incomplete BMP receptorkinase proten BRK-3 and an expression vector comprising a DNA sequencethat codes for an incomplete BMP type I receptor kinase protein.

As used herein, “human BMP receptor kinase protein-3” or “h-BRK-3” meansa protein having the amino acid sequence SEQ ID NO:2, as well asproteins having amino acid sequences substantially similar to SEQ IDNO:2, and which are biologically active in that they are capable ofbinding a BMP molecule (including, but not limited to BMP-2, DR-BMP-2,BMP-4, and/or BMP-7), or transducing a biological signal initiated by aBMP molecule binding to a cell, or crossreacting with antibodies raisedagainst h-BRK-3 protein, or peptides derived from the protein sequenceof h-BRK-3 or m-BRK-3 (see below), or forming a complex with a BMP typeI receptor, or co-immunoprecipitating with a BMP type I receptor whenantibodies specific for either h-BRK-3 or a BMP type I receptor areused.

As used herein, “truncated human BMP receptor kinase protein” or“h-BRK-3” means a protein having amino acid sequence SEQ ID NO:4, or asequence having the properties described above for BRK-3.

As used herein, “mouse BMP receptor kinase protein” or “m-BRK-3” means aprotein having amino acid sequence SEQ ID NO:8, or a sequence having theproperties described above for BRK-3.

As used herein, “BMP receptor kinase protein BRK-3” or “BRK-3” refersindividually and collectively to the receptor proteins h-BRK-3, t-BRK-3,and m-BRK-3 (and soluble and incomplete fragments thereof, describedabove, as well as BMP receptor kinase proteins substantially similar toh-BRK-3, t-BRK-3, and m-BRK-3 (and soluble and incomplete fragmentsthereof). Such receptor proteins, DNA sequences coding for the proteins,and recombinant expression vectors comprising said DNA are described andclaimed in U.S. Ser. No. 08/334,179, filed on Nov. 4, 1994, by Rosenbaumand Nohno.

As used herein, a “BMP Type I Receptor Kinase” is a protein capable ofbinding BMP-2, BMP-4 and/or other known BMPs, and bears sequencecharacteristics of a type I receptor including, but not limited to, anextracellular ligand bindingdomain containing a cysteine box and anupstream cysteine box, an SGSGSG motif, designated the GS domain, in theintracellular juxtamembrane region, an intracellular kinase domain thatis greater than about 85% similar to other type I receptors for otherligands in the TGF-β superfamily, and/or a relatively short carboxyterminus. As used herein, “BMP Type I Receptor Kinase” also includesreceptor proteins having the characteristics of a BMP type I receptor asdescribed in the literature, such as in: B. B. Koenig et al., Molecularand Cellular Biology, 14: 5961-5974 (1994); L. Attisano, et al.,Biochimica et Biophysica Acta, 1222: 71-80 (1994); J. Massagué, L.Attisano, and J. L. Wrana, Trends in Cell Biology, 4: 172-178 (1994);and ten Dijke, et al., J. Biological Chemistry, 269: 16985-16988 (1994).

Examples of BMP type I receptors include, but are not limited to: BRK-1(B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974(1994), the rat homologue of which is BMPR-Ia (K. Takeda, S. Oida, H.Ichijo, T. Iimura, Y. Maruoka, T. Amagasa, and S. Sasaki, Biochem.Biophys. Res. Communica., 204: 203-209 (1994)); BRK-2, also referred toas RPK-1 (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297-302(1993), and postulated to be the chicken homologue of ALK-6 (P. tenDijke, H. Yamashita, H. Ichijo, P. Franzén, M. Laiho, K. Miyazono, andC. -H. Heldin, Science, 264: 101-104 (1994)); ALK-2, which has beenshown to be a receptor for BMP-7 (ten Dijke et al., J. BiologicalChemistry, 269: 16985-16988 (1994)); the Xenopus BMP type I receptorthat binds BMP-2 and BMP-4 and which is involved in mesoderm induction(J. M. Graff, R. S. Thies, J. J. Song, A. J. Celeste, and D. A. Melton,Cell, 79: 169-179 (1994)); and type I receptors from Drosophila thatbind the decapentaplegic peptide, which is the Drosophila homologue ofBMP-2 and BMP-4. These Drosophila receptors are designated 25D1, 25D2,and 43E (T. Xie, A. L. Finelli, and R. W. Padgett, Science, 263:1756-1759 (1994); A. Penton, Y. Chen, K. Staehling-Hampton, J. L. Wrana,L. Attisano, J. Szidonya, J. A. Cassill, J. Massagué, and F. M.Hoffmann, Cell, 78: 239-250 (1994); and T. J. Brummel, V. Twombly, G.Marques, J. L. Wrana, S. J. Newfeld, L. Attisano, J. Massagué, M. B.O'Connor, and W. M. Gelbart, Cell, 78: 251-261 (1994)). Preferred BMPtype I receptors useful in the present invention include, but are notlimited to, polypeptides having the amino acid sequences substantiallysimilar to SEQ ID NO:12 (BRK-1), SEQ ID NO:16 (soluble BRK-1); SEQ IDNO:20 (incomplete BRK-1); SEQ ID NO:14 (BRK-2); SEQ ID NO:18 (solubleBRK-2); and SEQ ID NO:22 (incomplete BRK-2).

As used herein, “soluble fragment” refers to an amino acid sequencecorresponding to the extracellular region of BRK-1, BRK-2, or BRK-3which is capable of binding BMPs. Soluble fragments include truncatedproteins wherein regions of the receptor molecule not required for BMPbinding have been deleted. Examples of such soluble fragments for BRK-3include, but are not limited to, polypeptides having the amino acidsequences substantially similar to SEQ ID NO:6; SEQ ID NO:10; amino acidresidues 1-150 depicted in SEQ ID NO:2; amino acid residues 1-150depicted in SEQ ID NO:8; or polypeptides encoded by nucleic acidresidues substantially similar to SEQ ID NO:5; SEQ ID NO:9; nucleic acidresidues 409-858 depicted in SEQ ID NO:1, or nucleic acid residues17-466 depicted in SEQ ID NO:7.

Examples of soluble fragments for BRK-1 include, but are not limited to,polypeptides having the amino acid sequences substantially similar toSEQ ID NO:16; amino acid residues 1-152 in SEQ ID NO:12; polypeptidesencoded by nucleic acid residues substantially similar to SEQ ID NO:15;or polypeptides encoded by nucleic acid residues substantially similarto 11-466 in SEQ ID NO:11.

Examples of soluble fragments for BRK-2 include, but are not limited to,polypeptides having the amino acid sequences substantially similar toSEQ ID NO:18; amino acid residues 1-126 in SEQ ID NO:14; polypeptidesencoded by nucleic acid residues substantially similar to SEQ ID NO: 17;or polypeptides encoded by nucleic acid residues substantially similarto 355-732 in SEQ ID NO:13.

As used herein, “incomplete receptor kinase fragment” refers to an aminoacid sequence corresponding to the extracellular, transmembrane, andintracellular juxtamembrane region of BRK-1, BRK-2, or BRK-3 which iscapable of binding BMPs in a manner similar to the full-length receptor,but which is incapable of signalling due to deletion of theintracelllular kinase domain. Examples of such incomplete receptorfragments for BRK-3 include, but are not limited to, polypeptides havingthe amino acid sequences substantially similar to SEQ ID NO: 24; SEQ IDNO:26; amino acids 1-200 in SEQ ID NO:2; amino acids 1-200 in SEQ IDNO:8; polypeptides encoded by nucleic acid residues substantiallysimilar to SEQ ID NO: 23; polypeptides encoded by nucleic acid residuessubstantially similar to SEQ ID NO: 25; polypeptides encoded by nucleicacid residues substantially similar to 409-1008 in SEQ ID NO:1; orpolypeptides encoded by nucleic acid residues substantially similar to17-616 in SEQ ID NO:7.

Examples of incomplete receptor fragments for BRK-1 include, but are notlimited to, polypeptides having the amino acid sequences substantiallysimilar to SEQ ID NO:20; amino acid residues 1-231 in SEQ ID NO:12;polypeptides encoded by nucleic acid residues substantially similar toSEQ ID NO:19; or polypeptides encoded by nucleic acid residuessubstantially similar to 11-703 in SEQ ID NO:11.

Examples of incomplete receptor fragments for BRK-2 include, but are notlimited to, polypeptides having the amino acid sequences substantiallysimilar to SEQ ID NO:22; amino acid residues 1-201 in SEQ ID NO:14;polypeptides encoded by nucleic acid residues substantially similar toSEQ ID NO: 21; or polypeptides encoded by nucleic acid residuessubstantially similar to 355-957 in SEQ ID NO:13.

As used herein, a “BMP receptor kinase protein complex” is thecombination of a BMP type I receptor and BMP receptor kinase proteinBRK-3. The combination of the type I and BRK-3 receptors includes, butis not limited to, a combination of the type I and BRK-3 receptors insolution (e.g., as soluble fragments); a combination of the receptors(e.g., as soluble fragments) attached to a solid support; or acombination of the receptors (e.g., as full-length or incompletefragments) within a cell membrane of transfected cells.

As used herein, “digit-removed BMP-2” and “DR-BMP-2” refer to a fragmentof BMP-2 protein wherein the amino terminus of mature BMP-2 has beenremoved by mild trypsin digestion (B. B. Koenig et al., Molecular andCellular Biology, 14: 5961-5974 (1994)).

As used herein, “isolated”, in reference to the receptor protein of thepresent invention or DNA sequences encoding said protein, means that theprotein or DNA sequence is removed from the complex cellular milieu inwhich it naturally occurs, and said protein is expressible from said DNAsequence in a cell that does not naturally express it when operablylinked to the appropriate regulatory sequences.

As used herein, “substantially similar” when used to define either aminoacid or nucleic acid sequences, means that a particular subjectsequence, for example, a sequence altered by mutagenesis, varies from areference sequence by one or more substitutions, deletions, oradditions, the net effect of which is to retain biological activity ofthe BRK-3 protein. Alternatively, nucleic acid sequences and analogs are“substantially similar” to the specific DNA sequence disclosed herein ifthe DNA sequences, as a result of degeneracy in the genetic code, encodean amino acid sequence substantially similar to the reference amino acidsequence. In addition, “substantially similar” means a receptor proteinthat will react with antibodies generated against the BRK-3 protein orpeptides derived from the protein sequence of BRK-3.

As used herein, “biologically active” means that a particular moleculeshares sufficient amino acid sequence similarity with the embodiments ofthe present invention disclosed herein to be capable of bindingdetectable quantities of BMP-2 or BMP-4, or transmitting a BMP-2 orBMP-4 stimulus to a cell, for example, as a component of a hybridreceptor construct. Preferably, a biologically active BRK-3 receptorcomplex within the scope of the present invention means the receptorprotein kinase complex is capable of binding [¹²⁵I]-BMP-4 with nanomolaror subnanomolar affinity (K_(d) approximately equal to 10⁻⁹M).Preferably, the affinity is from about 1×10⁻¹²M to 1×10⁻⁹M, with aproportion of binding sites exhibiting a K_(d) less than 10⁻¹²M.

As used herein, “operably linked” refers to a condition in whichportions of a linear DNA sequence are capable of influencing theactivity of other portions of the same linear DNA sequence. For example,DNA for a signal peptide (secretory leader) is operably linked to DNAfor a polypeptide if it is expressed as a precursor which participatesin the secretion of the polypeptide; a promoter is operably linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operably linked to a coding sequence if it ispositioned so as to permit translation. Generally, operably linked meanscontiguous and, in the case of secretory leaders, contiguous in readingframe.

As used herein, “ATCC” means American Type Culture Collection,Rockville, Md.

As used herein, “bone morphogenetic protein 2” or “BMP-2” means apeptide encoded by a DNA sequence contained in ATCC No. 40345 (seeATCC/NIH REPOSITORY CATALOGUE OF HUMAN AND MOUSE DNA PROBES ANDLIBRARIES, sixth Edition, 1992, p. 57, hereinafter “ATCC/NIH REPOSITORYCATALOGUE”). Isolation of BMP-2 is disclosed in U.S. Pat. No. 5,013,649,Wang, Wozney and Rosen, issued May 7, 1991; U.S. Pat. No. 5,166,058,Wang, Wozney and Rosen, issued Nov. 24, 1992; and U.S. Pat. No.5,168,050, Hammonds and Mason, issued Dec. 1, 1992; each of which isincorporated herein by reference.

As used herein, “bone morphogenetic protein 4” or “BMP-4” means apeptide encoded by a DNA sequence contained in ATCC No. 40342 (seeATCC/NIH REPOSITORY CATALOGUE). Isolation of BMP-4 is disclosed in U.S.Pat. No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991,incorporated herein by reference.

As used herein, “bone morphogenetic protein 7” or “BMP-7” means apeptide encoded by a DNA sequence contained in ATCC No. 68020 and ATT68182 (see ATCC/NIH Repository Catalogue), where the cDNA in ATCC 68182is claimed to contain all of the nucleotide sequences necessary toencode BMP-7 proteins. Isolation of BMP-7 is disclosed in U.S. Pat. No.5,141,905, issued Aug. 25, 1992, to Rosen, et al., which is incorporatedherein by reference.

As used herein, “DNA sequence” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences (introns) which are typicallypresent in eukaryotic genes. Genomic DNA containing the relevantsequences could also be used. Sequences of non-translated DNA may bepresent 5′ or 3′ from the open reading frame, where the same do notinterfere with manipulation or expression of the coding regions. DNAsequences encoding the proteins provided by this invention can beassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

As used herein, “recombinant” means that a protein is derived from a DNAsequence which has been manipulated in vitro and introduced into a hostorganism.

As used herein, “microbial” refers to recombinant proteins made inbacterial, fungal (e.g., yeast), or insect expression systems.

As used herein, “recombinant expression vector” refers to a DNAconstruct used to express DNA which encodes a desired protein (forexample, BRK-3) and which includes a transcriptional subunit comprisingan assembly of 1) genetic elements having a regulatory role in geneexpression, for example, promoters and enhancers, 2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and 3) appropriate transcription and translation initiation andtermination sequences. Using methodology well known in the art,recombinant expression vectors of the present invention can beconstructed. Possible vectors for use in the present invention include,but are not limited to: for mammalian cells, pJT4 (discussed furtherbelow), pcDNA-1 (Invitrogen, San Diego, Calif.) and pSV-SPORT 1(Gibco-BRL, Gaithersburg, Md.); for insect cells, pBlueBac III orpBlueBacHis baculovirus vectors (Invitrogen, San Diego, Calif.); and forbacterial cells, pET-3 (Novagen, Madison, Wis.). The DNA sequence codingfor a BRK-3 protein receptor kinase of the present invention can bepresent in the vector operably linked to regulatory elements.

The present invention relates to a host cell co-transfected with anexpression vector comprising a DNA sequence that codes for BMP receptorkinase protein BRK-3 and an expression vector comprising a DNA sequencethat codes for a BMP type I receptor kinase protein. In one embodiment,the expression vector for the BRK-3 protein comprises a DNA sequencecoding for the h-BRK-3 receptor protein, or a soluble or incompletefragment thereof (The DNA can be genomic or cDNA.) Preferably theh-BRK-3 protein is coded for by the nucleic acid sequence SEQ ID NO: 1;the soluble fragment thereof is preferably coded for by the nucleic acidsequence SEQ ID NO: 5, the incomplete receptor fragment is preferablycoded for by nucleic acid SEQ ID NO:23. In another embodiment, theexpression vector for the BRK-3 protein comprises a DNA sequence codingfor the t-BRK-3 protein. (The DNA sequence can be genomic DNA or cDNA.)Preferably the DNA sequence is SEQ ID NO:3. In another embodiment, theexpression vector for the BRK-3 protein comprises a DNA sequence codingfor the m-BRK-3 protein, or a soluble or incomplete fragment thereof(The DNA sequence can be genomic DNA or cDNA.) Preferably the m-BRK-3protein is coded for by the DNA sequence SEQ ID NO:7; the solublefragment is preferably coded for by the DNA sequence SEQ ID NO:9; theincomplete fragment is preferably coded for by the DNA SEQ ID NO:25.

In a preferred embodiment of the present invention, the host cells ofthe present invention are co-transfected with the plasmid constructpJT6-mBRK-3L and the plasmid construct pJT4-J159F [BRK-1] or plasmidconstruct pJT3-BRK-2 [BRK-2], thereby resulting in co-expression ofm-BRK-3 and BRK-1, or m-BRK-3 and BRK-2, respectively. In anotherpreferred embodiment, the host cells of the present invention areco-transfected with the plasmid construct pJT6-mBRK-3 S and the plasmidconstruct pJT4-J159F [BRK-1] or plasmid construct pJT3-BRK-2 [BRK-2],thereby resulting in co-expression of m-BRK-3 and BRK-1, or m-BRK-3 andBRK-2, respectively. In another preferred embodiment, mammalian hostcells are co-transfected with the plasmid construct, pJT4-hBRK3T and theplasmid construct pJT6-J159F [BRK-1] or plasmid construct pJT3-BRK-2[BRK-2], thereby resulting in co-expression of t-BRK-3 and BRK-1, ort-BRK-3 and BRK-2, respectively. Transfection with the recombinantmolecules can be effected using methods well known in the art.

As used herein, “host cell” means a cell comprising a recombinantexpression vector described herein. Host cells may be stably transfectedor transiently transfected within a recombinant expression plasmid orinfected by a recombinant virus vector. The host cells includeprokaryotic cells, such as Escherichia coli, fungal systems such asSaccharomyces cerevisiae, permanent cell lines derived from insects suchas Sf-9 and Sf-21, and permanent mammalian cell lines such as Chinesehamster ovary (CHO) and SV40-transformed African green monkey kidneycells (COS).

In one embodiment, the present invention relates to a method that isuseful for identifying compounds capable of binding to a BMP receptorkinase protein. In another embodiment, the invention relates to a methodthat is useful for determining the concentration of a BMP receptorligand (e.g., BMP-2, BMP-4, or BMP-7, or another as-yet identified BMPreceptor ligand in a clinical sample. In each of these methods, a samplecomprising a putative ligand or a known ligand is introduced to a BMPreceptor kinase protein complex, wherein the receptor complex iscomprised of a BMP type I receptor kinase protein and BMP receptorkinase protein BRK-3. Preferably, the BRK-3 receptor kinase protein ish-BRK-3, having an amino acid sequence SEQ ID NO:2, or the solublefragment thereof having an amino acid sequence SEQ ID NO:6 or theincomplete fragment thereof having an amino acid sequence SEQ ID NO:24.Also preferred is the receptor protein m-BRK-3 having an amino acidsequence SEQ ID NO:8, or the soluble fragment thereof having an aminoacid sequence SEQ ID NO:10 or the incomplete fragment thereof having anamino acid sequence SEQ ID NO:26. Also preferred is the receptor proteint-BRK-3 having an amino acid sequence SEQ ID NO:4.

For example, BMP concentration in a sample can be determined byradioreceptor assay, in which unlabeled BMP in the sample competes withlabeled tracer BMP for binding to the BRK-3 receptor complex. As theamount of BMP in the sample increases, it reduces the amount of labeledBMP which is able to bind to the receptor protein complex comprisingBRK-3 and the type I receptor. Comparison with a standard curve preparedwith known concentrations of unlabeled BMP allows accurate quantitationof BMP concentration in the sample. Labeling of tracer BMP is preferablydone by iodination with [¹²⁵I]Nal. BRK-3 can be expressed in the outermembrane of a stable cell line which also expresses the BMP type Ireceptor kinase, or supplied as a soluble fragment in solution with asoluble type I receptor fragment, or as a soluble fragment covalentlyattached to a solid support in conjunction with a type I receptorcovalently attached to a solid support. To perform the assay, unlabeledBMP from the sample and labeled tracer BMP compete for binding to thereceptor until equilibrium is reached. The receptor-BMP complex is thenisolated from free ligand, for example by washing (in the case of anadherent cell line), rapid filtration or centrifugation (in the case ofa nonadherent cell line or receptor bound to a solid support), orprecipitation of the receptor-ligand complex with antibodies,polyethylene glycol, or other precipitating agent followed by filtrationor centrifugation (in the case of a soluble receptor). The amount oflabeled BMP in the complex is then quantitated, typically by gammacounting, and compared to known standards. These methods have beendescribed in the literature using other receptors (M. Williams, Med.Res. Rev., 11: 147-184 (1991); M. Higuchi and B. B. Aggarwal, Anal.Biochem., 204: 53-58 (1992); M. J. Cain, R. K. Garlick and P. M.Sweetman, J. Cardiovasc. Pharm., 17: S150-S151 (1991); each of which areincorporated herein by reference), and are readily adapted to thepresent BRK-3 receptor/BMP system. Such a radioreceptor assay can beused for diagnostic purposes for quantitation of BMP in clinicalsamples, where such quantitation is necessary.

The methods of the present invention is also useful in high-throughputscreens to identify compounds capable of binding to BRK-3, or ahomologous receptor protein, that is complexed to a BMP type I receptorkinase protein. In such a method, the higher the affinity of thecompound for the BRK-3/type I complex, the more efficiently it willcompete with the tracer for binding to the complex, and the lower thecounts in the receptor-ligand complex. In this case, one compares aseries of compounds within the same concentration range to see whichcompeted for receptor binding with the highest affinity.

This invention is useful for determining whether a ligand, such as aknown or putative drug, is capable of binding to and/or activating thereceptors encoded by the DNA molecules of the present invention.Transfection of said DNA sequence into the cell systems described hereinprovides an assay system for the ability of ligands to bind to and/oractivate the receptor complex encoded by the isolated DNA molecules.Recombinant cell lines, such as those described herein, are useful asliving cell cultures for competitive binding assays between known orcandidate drugs and ligands which bind to the receptor and which arelabeled by radioactive, spectroscopic or other reagents. Membranepreparations containing the receptor isolated from transfected cells arealso useful for competitive binding assays. Soluble receptors derivedfrom the ligand binding domain of the receptor can also be employed inhigh-throughput screening of drug candidates. Functional assays ofintracellular signaling can act as assays for binding affinity andefficacy in the activation of receptor function. In addition, therecombinant cell lines may be modified to include a reporter geneoperably linked to a response element such that a signal sent by thereceptor turns on the reporter gene. Such a system is especially usefulin high throughput screens directed at identification of receptoragonists. These recombinant cell lines constitute “drug discoverysystems”, useful for the identification of natural or syntheticcompounds with potential for drug development. Such identified compoundscould be further modified or used directly as therapeutic compounds toactivate or inhibit the natural functions of the receptor encoded by theisolated DNA molecule.

The soluble receptor protein complex of the present invention can beadministered in a clinical setting using methods such as byintraperitoneal, intramuscular, intravenous, or subcutaneous injection,implant or transdermal modes of administration, and the like. Suchadministration can be expected to provide therapeutic alteration of theactivity of the BMPs.

The nucleotide sequences disclosed herein, SEQ ID NO:3 and SEQ ID NO:1,represent the sequence of the DNA that codes for t-BRK-3 and h-BRK-3,respectively, isolated from human skin fibroblasts. SEQ ID NO:7represents the DNA sequence coding for m-BRK-3 receptor protein frommouse NIH3T3 cells. These sequences could be readily used to obtain thecDNA for BRK-3 from other species, including, but not limited to, rat,rabbit, Drosophila, and Xenopus. These sequences could also readily beused to obtain the cDNA for other BMP type II receptors from mouse andother species listed above which are capable of binding BMPs in acomplex with the type I receptors. These cDNA sequences can also bereadily used to isolate the genomic DNA for BRK-3. This would permitanalysis of the regulatory elements controlling receptor geneexpression, which may offer new opportunities for therapeuticintervention and disease diagnosis. The nucleotide sequences are alsouseful to determine the distribution of the BRK-3 receptor in normaltissues and in disease states, which allows an assessment of itsphysiological role in vivo.

The present invention further relates to a method for determiningwhether a test compound produces a signal upon binding to a BMP receptorprotein complex. Such a method comprises employing the BMP receptorprotein complex in a protein phosphorylation assay. Proteinphosphorylation assays are generally known in the art. Hardie, D. G.,“Protein Phosphorylation A Practical Approach”, IRL Press: Oxford, N.Y.,Tokyo, (1993), incorporated herein by reference, provides a generaloverview of phosphorylation assays. The method for determining whether atest compound produces a signal upon binding to the BMP receptor protiencomplex comprises (a) labeling BMP receptor protein complex expressingcells with ³²P, wherein the cells have been transformed with a DNAsequence coding for BMP receptor kinase protein BRK-3 and a DNA sequencecoding for a BMP type I receptor kinase protein; (b) culturing (i) afirst set of the cells in the presence of the test compound, and (ii) asecond set of the cells in the absence of the test compound; (c)quantitating via autoradiography any phosphorylated proteins producedfrom step (b); and (d) comparing the amount of phosphorylated proteinsquantitated in step (c) from the first set of cells to the amount ofphosphorylated proteins quantitated in step (c) for the second set ofcells.

For purposes of illustrating a preferred embodiment of the presentinvention, the following non-limiting examples are discussed in detail.

EXAMPLE 1 Generation of PCR Fragments

In order to generate a PCR fragment of type II receptors related to theTGF-β type II receptor, primers shown in FIG. 1 are designed from thekinase domains of the TGF-β type II receptor. For the first round ofPCR, the primers are TSK-1, derived from kinase domain II, and TSK-2,derived from kinase domain VIII. The template DNA consists of cDNAprepared from mRNA isolated from human skin fibroblasts from a 9 monthold male. The PCR reaction, carried out in a total volume of 50 μl,contains approximately 0.2 μg of this cDNA, primers TSK-1 and TSK-2 at aconcentration of 15 μM, stocks of all four deoxynucleotides at aconcentration of 0.2 mM each, 1.5 unit of DNA polymerase from Thermusthermophilus (hereafter, Tth polymerase) (Toyobo, Osaka, Japan) andreaction buffer for the Tth polymerase (Toyobo, Osaka, Japan). After aninitial melting period of 1 min at 94° C., the temperature cycle iscarried out as follows for 35 cycles: melting, 92° C. for 40 sec;annealing, 48° C. for 40 sec; extension, 75° C. for 90 sec. After the35th cycle, the reaction is held at 75° C. for an additional 5 min tocomplete the extension.

Several bands are amplified, including some in the area of 470 basepairs (bp) corresponding to the predicted sequence length of a type IIreceptor homologous to the TGF-β type II receptor. Accordingly,fragments in this size range are recovered from an agarose gel usingQIAEX (Qiagen, Chatsworth, Calif.; a kit for gel purification of DNAfragments, including activated silica spheres and buffers) according tothe manufacturer's instructions, then resuspended in 10 mM Tris, pH 8.0,1 mM EDTA (TE) in a volume of 20 μl.

To reduce the background from fragments amplified from cDNAs not relatedto the TGF-β type II receptor, a second round of PCR is carried outusing “nested” primers based on conserved regions of the TGF-β type IIreceptor located within the 470 bp region amplified in the first round.The nested primers are AVR-5, derived from kinase domain IV of theTGF-J3 type II receptor, and TSK-4, derived from kinase domain VIB (FIG.1). The template consists of an aliquot (0.5 μl) of the PCR fragmentsisolated from the first round of PCR. To this is added the primers AVR-5(5 μM) and TSK-4 (15 μm), all four deoxynucleotides (0.2 mM each), 1.5units of Tth DNA polymerase, and reaction buffer for the Tth DNApolymerase, in a total volume of 50 μl. The temperature cycle program isexecuted exactly as described above for the first round of PCR. Agarosegel electrophoresis of the PCR reaction products shows amplification ofa band in the range of 300 bp, as expected. This fragment is isolatedusing QIAEX.

In order to subclone the PCR product of the second PCR reaction, thepurified fragment is phosphorylated using polynucleotide kinase andligated to the cloning vector pGEM7Zf (+) (Promega, Madison, Wis.) whichhas previously been cut with Sma I and dephosphorylated. The ligationmix is used to transform E. coli XL 1-Blue (Stratagene, La Jolla,Calif.). When the transformation mix is plated on agar containingisopropyl-β-D-thiogalactoside (IPTG) and5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), colonies areobtained which lack blue color, indicating the presence of an insert.Plasmid DNA is prepared from a selection of these colonies. Three of thecandidate plasmids, designated HSK7-1, HSK7-2, and HSK7-4 are found tohave inserts of the expected size (300 bp). Upon sequencing of theinserts, the 300 bp insert from HSK7-2 is found to encode a portion of anovel kinase that is predicted to be a novel member of the TGF-βreceptor superfamily. Accordingly, the HSK7-2 PCR fragment is used as aprobe to isolate the full-length receptor clone.

EXAMPLE 2 Isolation of Human t-BRK-3 cDNA

In order to locate the cDNA corresponding to the 300 bp insert inHSK7-2, a cDNA library is constructed from the same mRNA used to isolatethe PCR fragment. This is accomplished using the SUPERSCRIPT ChoiceSystem (Life Technologies, Gaithersburg, Md.; a kit for cDNA synthesis,including primers, adapters, SUPERSCRIPT II RNAse H⁻ ReverseTranscriptase (Life Technologies, Gaithersburg Md.; a modified form ofreverse transcriptase from Moloney murine leukemia virus), enzymes,nucleotides, buffers, and gel filtration columns) according to themanufacturer's instructions, except that 180 units of RNase inhibitor(Takara, Kyoto, Japan) is added to the first strand synthesis. Thetemplate is mRNA (4 μg) from human skin fibroblasts from a 9 month oldmale. A total of 4 μg of cDNA is obtained after first and second strandsynthesis. This is followed by the addition of Eco RI adapters (suppliedwith the kit) which contain internal Not I and Sal I sites. The EcoRI-adapted cDNA is then phosphorylated and subjected to sizefractionation according to the manufacturer's instructions, using gelfiltration columns provided with the kit.

The size fractionated cDNA is ligated into the Eco RI site of the phageλgt10, and packaged in vitro with GIGAPACK II Gold Packaging Extract(Stratagene, La Jolla, Calif.; a restriction-minus in vitro packagingextract for high-efficiency construction of cDNA libraries in λ phage)according to the manufacturer's instructions. A total of 8.1×10⁵ phagesare obtained.

The library is screened on ten HYBOND Nylon membranes (Amersham,Arlington Heights, Ill.; nylon membranes optimized for immobilization ofnucleic acids), at a density of 1×10⁵ plaques/filter. The insert fromHSK7-2 is labeled with the MULTIPRIM DNA Labeling System (Amersham,Arlington Heights, Ill.; a kit for random primer labeling of DNA,including Klenow DNA polymerase, primers, and buffers) according to themanufacturer's instructions. The labeled probe is allowed to hybridizeto the library filters in 50% formamide, 6× SSPE (1×SSPE=0.14 M NaCl, 8mM sodium phosphate, 0.08 mM EDTA, pH 7.7), 5× Denhardt's solution(1×Denhardt's=0.02% Ficoll type 400, 0.02% polyvinylpyrrolidone, 0.02%BSA), 0.5% sodium dodecyl sulfate (SDS), and 100 μg/ml denatured salmonsperm DNA at 42° C. for 12 hr. The blot is then washed in 2× SSPE, 0.1%SDS three times at room temperature (15 minutes each), followed by a 1hr wash at 42° C.

After three rounds of screening, 3 independent clones are obtained. Oneof the clones, designated HSK723, is found to encode the same sequenceas the HSK7-2 insert. Complete DNA sequence is obtained for this clone.The cDNA from this clone is designated t-BRK-3.

EXAMPLE 3 t-BRK-3 Sequence Analysis

The DNA sequence of this t-BRK-3 clone is shown in SEQ ID NO:3, and thededuced protein sequence of t-BRK-3 in SEQ ID NO:4. The t-BRK-3 openreading frame derived from clone HSK723 encodes a protein of at least583 amino acids. No stop codon is observed to be located in-frame in the3′ region of the HSK723 cDNA, indicating that this clone is incompleteat the 3′ end. It is thus designated t-BRK-3.

The deduced protein sequence of t-BRK-3 shown in SEQ ID NO:4 is searchedagainst all translated protein sequences in GenBank Release 84.0, datedAug. 15, 1994, using a standard Needleman-Wunsch algorithm (S. B.Needleman and C. D. Wunsch, J. Mol. Biol. 48: 443-453 (1970)), and isfound to represent a novel sequence.

Analysis of the predicted protein sequence reveals a predicted structureof a TGF-β type II superfamily member transmembrane serine/threoninekinase. The predicted single transmembrane region encompasses residues151-172 in SEQ ID NO:4. Three potential N-linked glycosylation sites arelocated at amino acid residues 55, 110, and 126 in the predictedextracellular domain. Amino acids 116-123 in SEQ ID NO:4 contain thecluster of cysteine residues called the “cysteine box” that is acharacteristic of receptors for ligands of the TGF-β superfamily. Thecysteine box of t-BRK-3 is identical in 6 of 8 amino acid residues tothe cysteine box of the DAF-4 type II receptor for BMP-2 and BMP-4.However, the overall sequence identity of t-BRK-3 to DAF-4 in theextracellular domain is only 7.1%.

Amino acids 200-504 (in SEQ ID NO: 4) in the predicted cytoplasmicregion of t-BRK-3 contains all of the consensus sequences thatcharacterize a protein kinase domain with predicted specificity forserine/threonine residues (S. K. Hanks, A. M. Quinn, and T. Hunter,Science, 241: 42-52 (1988)).

EXAMPLE 4 Construction of Expression Vectors for t-BRK-3, BRK-1, BRK-2,and DAF-4

In order to express t-BRK-3 in mammalian cells, it is subcloned into thevector pJT4, designed for transient expression. The pJT4 vector,optimized for transient expression in COS cells, includes thecytomegalovirus early promoter and enhancer, which gives very efficienttranscription of message; an “R” element from the long terminal repeatof the human T-cell leukemia virus-1, which has been shown to increaseexpression levels further; an intron splice site from SV40, which isbelieved to enhance message stability; a multiple cloning site; apolyadenylation signal derived from SV40, which directs the addition ofa poly A tail to the message, as is required for most eukaryotic mRNA;and the SV40 origin of replication, which permits the replication of theplasmid to extremely high copy number in cells which contain the SV40large T antigen, such as COS cells. In addition, for manipulation andamplification of the vector in bacteria, the vector contains an E. coliorigin of replication and an ampicillin resistance gene. Insertion ofthe truncated human BRK-3 cDNA into pJT4 is accomplished as follows.

Since no stop codon had been identified in the 3′ region of the kinasedomain, PCR is performed to insert a stop codon to permit translation ofthe protein. Accordingly, a PCR primer is designed to insert two stopcodons after nucleotide 2028 in SEQ ID NO: 3, thus terminating thekinase after lie 540 in SEQ ID NO: 4. This is chosen to correspond tothe length of the activin type II receptor (L. S. Mathews and W. V.Vale, Cell, 65: 973-982 (1991)), so that it should be sufficient forproper folding of the kinase domain. The stop codons are followed by aKpn I site. The complete sequence of the primer (which includes thereverse complement of nucleotides 2013-2028 in SEQ ID NO:3) is 5′ ACGCGG TAC CTC ACT AAA TTT TTG GCA CAC GC 3′. A second primer is designedas an exact match to the t-BRK-3 sequence in the area of the Afl IIIsite (nucleotides 1618-1637 in SEQ ID NO:3), having the sequence 5′ GTAGAC ATG TAT GCT CTT GG 3′. The template for the reaction is cloneHSK723, described in example 2, which contains the cDNA for t-BRK-3 inBLUESCRIPT II SK (+) (Stratagene, La Jolla, Calif.; a 2.96 kbcolony-producing phagemid derived from pUC 19).

PCR is carried out using the GENE AMP PCR Kit with AMPLITAQ DNAPolymerase (Perkin Elmer, Norwalk, Conn.; a kit containing componentsnecessary for amplification of DNA using the polymerase chain reaction,including AMPLITAQ, a recombinant modified form of the DNA polymerasefrom Thermus aquaticus (Perkin-Elmer, Norwalk Conn.), nucleotides, andbuffers), according to the manufacturer's instructions, using a GENE AMPPCR System 9600 Thermocycler (Perkin Elmer, Norwalk, Conn.). An initialmelting at 95° C. for 5 min is followed by 20 cycles of the followingprogram: melting at 95° C. for 1 min, annealing at 50° C. for 1 min, andextension at 72° C. for 1 min. After the last cycle, the temperature isheld at 72° C. for an additional 2 min to complete extension.

The resulting amplified band, at the expected size of 400 bp, isisolated from an agarose gel and digested with Afl III and Kpn I.Meanwhile, the cDNA for t-BRK-3 is digested with Eco RV and Afl III, andthe vector pJT4 is digested with Eco RV and Kpn I. These three isolatedfragments are ligated in a single step to give the constructpJT4-hBRK3T, shown in FIG. 2. To confirm that no errors are introducedduring PCR, the region from the Afl III site to the KpnI site at the 3′end is sequenced using the TAQ DYE DEOXY Terminator Cycle Sequencing Kit(Applied Biosystems, Foster, Calif.; kit containing components forautomated DNA sequencing using the dideoxy terminator method, includingAMPLITAQ, nucleotide mix, dye-labeled dideoxy nucleotide terminators,and buffers) and an Applied Biosystems Model 373A Automated DNASequencer. No errors are found.

To determine the effects of co-expression of t-BRK-3 with type I BMPreceptors, it is necessary to co-express the cDNA for t-BRK-3 with thecDNA for BRK-1 or the cDNA for BRK-2. The DNA sequence for mouse BRK-Iis shown in SEQ ID NO: 11, and the deduced amino acid sequence for mouseBRK-1 is shown in SEQ ID NO: 12. The DNA sequence for chicken BRK-2 isshown in SEQ ID NO: 13, and the deduced protein sequence shown forchicken BRK-2 is shown in SEQ ID NO: 14.

For mammalian expression of BRK-1, the plasmid pJT4-J159F is used.Construction of this plasmid is described in U.S. Ser. No. 08/158,735,filed Nov. 24, 1993 by Cook, et al. and B. B. Koenig et al., Molecularand Cellular Biology 14: 5961-5974 (1994); ATCC 69457. Briefly, theconstruct containing the BRK-1 cDNA subcloned in BLUESCRIPT SK (−) islinearized with the restriction endonuclease Alf III and the overhangingend filled in using DNA Polymerase I Klenow fragment. The linearizedplasmid is then digested with Not I, liberating the insert from theplasmid. The insert is then subcloned into the pJT4 expression vector atthe Not I and EcoRV sites. The blunt end generated by the Klenowreaction is compatible with the EcoRV site, which is also a blunt end;ligation eliminates the Eco RV site. The construct pJT4-J159F is shownin FIG. 3.

For mammalian expression of BRK-2, its cDNA is subcloned into the vectorpJT3. This vector is identical to pJT4, described in this example,except that the multiple cloning site is in the opposite orientation,and an additional Not I site is present at the 5′ end of the multiplecloning site. The cDNA for BRK-2 (see S. Sumitomo, et al., DNA Sequence3: 297-302 (1993)), originally obtained in the vector pRc/CMV(Invitrogen, San Diego, Calif.; a mammalian expression vector), isexcised by digestion with Kpn I and Xho I. It is subcloned into pJT3 atthe Kpn I and Sal I sites. This regenerates a Kpn I site at the 5′ endof BRK-2, while the Xho I and Sal I sites are destroyed. The resultingconstruct is designated pJT3-BRK-2 and is shown in FIG. 4.

For mammalian expression of DAF-4, the type II BMP receptor fromCaenorhabditis elegans (M. Estevez, L. Attisano, J. L. Wrana, P. S.Albert, J. Massagué, and D. L. Riddle, Nature, 365: 644-9 (1993), thecDNA is obtained in BLUESCRIPT II and subcloned into pJT4 as follows. A2.4 kb fragment containing the daf-4 cDNA is excised by digestion withDra I and Apa I. This fragment is subcloned into pJT4 at the Sma I andApa I site. The Apa I site is regenerated, while the Dra I and Sma Isites are destroyed. This construct is designated pJT4-Daf4, and isshown in FIG. 5.

For mammalian expression of m-BRK-3, see Example 10, below.

EXAMPLE 5 Mammalian Expression of t-BRK-3, BRK-1, BRK-2, and DAF-4

Transient expression of BRK-3 in mammalian cells using pJT4-hBRK3T iscarried out in COS-7 cells (ATCC CRL 1651) using electroporation orCOS-1 cells (ATCC CRL 1650) using DEAE Dextran (Pharmacia Biotech,Piscataway, N.J.).

COS-7 cells are grown to confluence in Dulbecco's Modified Eagle (DME)high glucose media supplemented with 10% fetal bovine serum (Hyclone,Logan, Utah), nonessential amino acids (GIBCO, Gaithersburg, Md.), andglutamine, then trypsinized to release cells from the plate. Thedetached COS-7 cells are pelleted in a tabletop centrifuge, thenresuspended in fresh media at a concentration of 6.25×10⁶ cells/ml. Thecell suspension (5×10⁶ cells, 0.8 ml) is transferred to the cuvette of aBioRad GENE PULSER electroporation system (BioRad, Hercules, Calif.).The purified plasmid containing the receptor DNA of interest (10 μg forpJT4-J159F and pJT3-BRK2 and/or 20 μg for pJT4-hBRK3T) is added to thecuvette, and the cells subjected to electoporation at 4.0 kV/cm, with acapacitance of 25 μFd. Cells are then plated (400,000 cells per well for12 well plates and 5×10⁶ cells for 100 mm plates) and allowed torecover. Fresh media is supplied after 24 hr. At 48 hr, cells are readyto be tested for binding of BMP-4.

For transient expression of BMP receptors in COS-1 cells, the cells aregrown to approximately 50%-80% confluence in DME high glucose mediasupplemented with 10% fetal bovine serum (HyClone, Logan, Utah),nonessential amino acids, and glutamine in 100 mm plates. The cells arewashed twice with 37° C. serum-free DME media, after which 4 ml of DNAmixture is added to each 100 mm plate. The DNA mixture contains DME, 10%Nu-Serum (Collaborative -Biomedical Products, Bedford, Mass.), 400 μg/mlDEAE-Dextran (Pharmacia, Piscataway, N.J.), 0.1 mM chloroquine (Sigma,St. Louis, Mo.), and the cDNAs of interest: for t-BRK-3, 16 μgpJT4-hBRK3T; for BRK-1, 8 μg pJT4-J159F; for BRK-2, 8 μg pJT3-BRK; forDAF-4, 16 μg pJT4-Daf4. The cells are then incubated at 37° C. with theDNA mixture for 3 hr. The solution is aspirated and the cells areincubated with 4 ml of a solution containing 10% dimethylsulfoxide(DMSO) in Dulbecco's phosphate buffered saline without calcium ormagnesium (PBS; Life Technologies, Gaithersburg, Md.). After 2 min, theDMSO solution is aspirated, the cells are washed with the growth mediadescribed above, and fresh media is returned to the plates. Thetransfected cells are split into 12 well plates 24 hr post transfectionfor whole cell binding or cross linking. After 48 to 68 hr the cells aresuitable for binding analysis.

EXAMPLE 6 Generation of the Radiolabeled BMP-4 Ligand

[¹²⁵I]-BMP-4 is prepared using IODOBEADS (Pierce, Rockford, Ill.;immobilized chloramine-T on nonporous polystyrene beads). LyophilizedBMP-4 (2 μg) is taken up in 50 μl of 10 mM acetic acid and added to 450μl of phosphate-buffered saline (PBS) (Sigma, St. Louis, Mo.) on ice. Tothe tube is added 500 μCurie of ¹²⁵I (Amersham, Arlington Heights, Ill.)(2200 Ci/mmol) in 5 μl, and one IODOBEAD. The reaction is incubated onice for 10 min with occasional shaking. The reaction is then terminatedby removal of the reaction from the IODOBEAD. To remove unreacted ¹²⁵I,the mixture is applied to a PD-10 gel filtration column (Pharmacia,Piscataway, N.J.) previously equilibrated in 10 mM acetic acid, 0.1 MNaCl, 0.25% gelatin. The resulting labeled protein is >95% precipitableby trichloroacetic acid, indicating that all ¹²⁵I is protein bound, andhas a typical specific activity of 4000 to 9000 Ci/mmol.

Alternatively, BMP-4 is labeled with ¹²⁵I by the chloramine-T method (C.A. Frolik, L. M. Wakefield, D. M. Smith, and M. B. Sporn, J. Biol.Chem., 259: 10995-11000 (1984)). BMP-4 (2 μg) is taken up in 5 μl of 30%acetonitrile, 0.1% trifluoracetic acid (TFA) plus an additional 5 μl of1.5 M sodium phosphate, pH 7.4. Carrier free ¹²⁵I (1 mCi, 9 μl) isadded, together with 2 μl of a chloramine T solution (100 μg/ml). Anadditional 2 μl of the chloramine T solution is added at 2.0 min and at3.5 min. After 4.5 minutes, the reaction is stopped by the addition of10 μl of 50 mM N-acetyl tyrosine, 100 μl of 60 mM potassium iodide, and100 μl of 11M urea, 1 M acetic acid. After a 3.5 minute incubation,unreacted iodine is removed on a PD-10 gel filtration column (Pharmacia,Piscataway, N.J.) run in 4 mM HCl, 75 mM NaCl, 1 mg/ml bovine serumalbumin (BSA). The resulting labeled protein is >95% precipitable bytrichloroacetic acid, indicating that all ¹²⁵I is protein bound, and hasa typical specific activity of 3000-8000 Ci/mmol.

EXAMPLE 7 Characterization of BMP-4 Binding to t-BRK-3

Binding of BMP-4 to t-BRK-3 can be demonstrated by whole cell binding ofradiolabeled BMP-4, and by covalent crosslinking of radiolabeled BMP-4to the receptor. These two methods are described in detail below.

a. Whole Cell Binding:

COS-7 or COS-1 cells are transfected with pJT4-hBRK3T as described inexample 5. After transfection, cells are seeded into 12 well plates andthe binding experiments are carried out at 48 to 68 hr. At that time,cells are washed once with binding buffer (50 mM HEPES, pH 7.4, 128 mMNaCl, 5 mM KCl, 5 mM MgSO₄, 1.2 mM CaCl₂, 2 mg/ml BSA), thenequilibrated in the same buffer at 4° C. for 30-60 min with gentleshaking. The buffer is then aspirated, and to each well is added 500 μlof binding buffer (4° C.), containing [¹²⁵I]-BMP-4 tracer (100-400 pM),as well as varying concentrations of unlabeled BMP-2, BMP-4, or otherunlabeled ligand, depending on the assay. For determination ofnonspecific binding, BMP-4 is added to the binding buffer at a finalconcentration of 10 to 50 nM. To prevent degradation of ligand duringthe incubation, a protease inhibitor cocktail is also added, to give afinal concentration of 10 μg/ml leupeptin, 10 μg/ml antipain, 50 μg/mlaprotinin, 100 μg/ml benzamidine, 100 μg/ml soybean trypsin inhibitor,10 μg/ml bestatin, 10 μg/ml pepstatin, and 300 μM phenylmethylsulfonylfluoride (PMSF). The cells are incubated for 4 hr at 4° C. with gentleshaking. At the end of the incubation period, the buffer is aspirated,and the cells are rinsed 4 times with 1 ml washing buffer (50 mM HEPES,pH 7.4, 128 mM NaCl, 5 mM KCl, 5 mM MgSO₄, 1.2 mM CaCl₂, 0.5 mg/ml BSA).After the final wash is aspirated, 200 μl of solubilization buffer (10mM Tris Cl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100) is added to eachwell and incubated at room temperature for 15-30 min. The solubilizedcells are then transferred to fresh tubes and counted in a Packard Model5005 COBRA Gamma Counter (Packard Instruments, Meriden, Conn.).

Results are shown in FIG. 6, which shows specific binding of[¹²⁵I]-BMP-4 to NIH3T3 cells (ATCC CRL 1658), which display significantendogenous binding of BMP-4, and COS 7 cells transfected with the cDNAfor t-BRK-3 in the presence and absence of BRK-1 and BRK-2. t-BRK-3 iscapable of binding [¹²⁵I]-BMP-4 when expressed alone (bar on far right),at a level similar to that seen for BRK-1 and BRK-2 expressed alone.Binding of [¹²⁵I]-BMP-4 is increased by co-expression of t-BRK-3 withBRK-1, and to a greater extent by co-expression of t-BRK-3 with BRK-2.

b. Covalent Crosslinking:

Bifunctional crosslinking reagent disuccinimidyl glutarate (DSG)(Pierce, Rockford, Ill.) is used to covalently crosslink boundradiolabeled ligand to its receptor by reaction with free amino groupson lysine residues in the two proteins. Following the crosslinking,cellular proteins are separated by gel electrophoresis, and radioactivebands visualized. The labeled bands represent the receptor selectively“tagged” with the radiolabeled ligand. In this procedure, cells aretransfected with the cDNA for BRK-3, and/or BRK-1 or BRK-2, as describedin example 5, then seeded into 12 well plates. At 48-68 hr aftertransfection, the cells are washed, equilibrated, and incubated with[¹²⁵I]-BMP-4 and competing unlabeled ligands as described in thisexample for whole cell binding studies. After completion of the 4 hrincubation with ligand, the cells are washed two to three times at 4° C.with 2 ml of binding buffer having the same composition as describedabove, except that no BSA is added. To each well is then added 1 ml offresh BSA-free binding buffer, followed by freshly prepared DSG to afinal concentration of 135 μM. After swirling gently to mix the DSG, theplates are incubated for exactly 15 minutes at 4° C. with gentleshaking. At this point the media is aspirated and the cells washed with3 ml detachment buffer (10 mM Tris base, 0.25 M sucrose, 1 mM EDTA, 0.3mM PMSF) or PBS. Solubilization buffer (50 μl) is then added to eachwell and the cells are allowed to solubilise for 30-45 minutes at 4° C.with shaking. An aliquot of the sample (20 μl) is transferred to a freshtube and 5 μL of 5× sample loading buffer (0.25 M TrisCl, pH 6.8, 10%SDS, 0.5 M DTT, 0.5% bromophenol blue, 50% glycerol; purchased from FivePrime Three Prime, Boulder, Colo.) is added. The samples are boiled for5 min and centrifuged (13,0000×g, 5 min). The supernatants are loadedonto 7.5% SDS-polyacrylamide gels (Integrated Separation Systems,Natick, Mass.) and subjected to electrophoresis. The gels are stained in0.12% Coomassie Blue R250, 5% methanol, 7.5% acetic acid; destained in5% methanol, 7.5% acetic acid; then dried. Radioactivity on the driedgel is visualized and quantitated on a PHOSPHORIMAGER (MolecularDevices, Sunnyvale, Calif., a device for quantitation of radioactivityusing stable phosphor screens), or subjected to autoradiography usingKodak X-OMAT AR autoradiography film (Kodak, Rochester, N.Y.).

Results are shown in FIG. 7. When t-BRK-3 is expressed alone in COS-1cells, no crosslinked band is seen. Expression of BRK-1 alone results ina crosslinked band at a molecular weight of 78 kD, corresponding to thepredicted molecular weight of BRK-1 plus the monomer molecular weight ofBMP-4. Co-expression of t-BRK-3 and BRK-1 results in the appearance of aband of similar size to that for BRK-1, as well as a new crosslinkedband at 94 kD, corresponding to the predicted molecular weight oft-BRK-3 plus the monomer molecular weight of crosslinked BMP-4.Similarly, expression of BRK-2 alone yields a single crosslinked band at75 kD, corresponding to the predicted molecular weight of BRK-2 plus thecrosslinked BMP-4 monomer. Co-expression of t-BRK-3 with BRK-2 yields acrosslinked band corresponding to that seen for BRK-2 alone, as well asa new crosslinked band at 94 kD, again corresponding to the predictedmolecular weight of t-BRK-3 plus the monomer molecular weight ofcrosslinked BMP-4. Thus, crosslinking of [¹²⁵I]-BMP-4 to t-BRK-3 isobserved only in the presence of a co-expressed type I BMP receptor.

EXAMPLE 8 Demonstration of Complex Formation with Type I BMP Receptors

Receptors of the TGF-β receptor family have been shown to form complexesinvolving a type I and a type II receptor (L. Attisano, J. L. Wrana, F.Lopez-Casillas, and J. Massagué, J. Biochim Biophys. Acta, 1222: 71-80(1994)). In order to demonstrate that the type II BMP receptor t-BRK-3can form a complex with the type I BMP receptors BRK-1 and BRK-2, COS-1cells are co-transfected with the cDNA for t-BRK-3 and BRK-1, or t-BRK-3and BRK-2, as described in Example 5. The receptors are crosslinked to[¹²⁵I]-BMP-4, then subjected to immunoprecipitation with antibodiesspecific for the type I receptors BRK-1 and BRK-2. If antibodiesspecific for a type I receptor precipitate not only the type I receptorcrosslinked to [¹²⁵I]-BMP-4, but also BRK-3 crosslinked to [¹²⁵I]-BMP-4,this indicates that the two receptors must be forming a complex, asexpected for type I and type II receptors having the same ligand-bindingspecificity.

Antibodies specific for the type I receptors BRK-1 and BRK-2 aregenerated using as antigen the peptide LNTRVGTKRYMAPEVLDESLNKNC (SEQ IDNO: 27) (B. B. Koenig, et al., Molec. Cell. Biol., 14: 5961-5974(1994)). This peptide is based on the amino acid sequence of BRK-1 inthe intracellular kinase domain, amino acids 398-420 in SEQ ID NO: 12,with the addition of a cysteine at the C terminus to permit conjugationof the peptide. Comparison of the amino acid sequence of the kinasedomain of BRK-1 with the kinase domain of the Raf protein suggests thatthis region of BRK-1 corresponds to a region of the Raf kinase which wasused to make highly specific antibodies (W. Kolch, E. Weissinger, H.Mischak, J. Troppmair, S. D. Showalter, P. Lloyd, G. Heidecker, and U.R. Rapp, Oncogene, 5: 713-720 (1990)). This peptide is conjugated bystandard methods to keyhole limpet hemocynanin, and used to immunizethree New Zealand White rabbits (Hazleton Washington, Vienna, Va.). Theresulting antisera are evaluated for their ability to recognize theoriginal peptide coated on plastic, using an antibody capture ELISA. Theantisera are designated 1378, 1379, and 1380. These antibodies are shownto immunoprecipitate BRK-1 from COS-7 cells transfected with the cDNAfor BRK-1, using the procedure detailed in this example (B. B. Koenig,et al., Mol. Cell Biol., 14: 5961-5974 (1994)). Because the sequence ofBRK-2 is nearly identical to that of BRK-1 in this region, theseantibodies are subsequently tested for their ability toimmunoprecipitate BRK-2 as well, and are found to be effective for thispurpose. Antibody 1379 gives superior results for immunoprecipitation ofBRK-1, and antibody 1380 is preferred for immunoprecipitation of BRK-2.

In the immunoprecipitation procedure, COS-7 or COS-1 cells aretransfected with the cDNA for t-BRK-3 and/or BRK-1, BRK-2, or DAF-4 asdescribed in Example 5, and plated into 100 mm dishes. They are thencrosslinked to [¹²⁵I]-BMP-4 as described in example 7, except that theincubation with [¹²⁵I]-BMP-4 and unlabeled ligand is carried out in atotal of 4 ml, instead of 500 μl, and all other volumes are increasedaccordingly. Following the crosslinking, cells are washed three timeswith ice-cold PBS, then lysed with 1 ml of RIP buffer (20 mM TrisCl, pH8.0, 100 mM NaCl, 1 mM Na₂EDTA, 0.5% Nonidet P-40, 0.5% sodiumdeoxycholate, 10 mM sodium iodide, and 1% bovine serum albumin) for 10min. The lysate is centrifuged in a microcentrifuge at 13,000 rpm for 10min at 4° C. The supernatant is transferred to a fresh tube and made0.1% in SDS. To remove any existing antibody present in the lysate, 50μl of PANSORBIN (Calbiochem, La Jolla, Calif.; a 10% solution ofStaphylococcus aureus) is added. After a 30 minute incubation at 4° C.,the lysate is centrifuged as before, and the supernatant againtransferred to a fresh tube.

The primary antibody-1379 when cells are transfected with t-BRK-3 andBRK-1; 1380 when cells are transfected with t-BRK-3 and BRK-2-is thenadded to the tube at a final dilution of 1:100, and incubated for 2 hron ice or overnight at 4° C. To precipitate the complex ofantigen:primary antibody, 25-50 μl of PANSORBIN is then added andincubated 30 min on ice. The complex is pelleted at 13,000 rpm for 10min in a microcentrifuge and the supernatant discarded. The pellet iswashed twice in RIP buffer containing 0.1% SDS, and once in TNEN buffer(20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40). The pellet isresuspended in 25 μl of 1× sample loading buffer. (Alternatively, thepellet may be washed twice with TNEN buffer, with similar results.) Thesample is boiled for 5 min, centrifuged for 5 min, and subjected to gelelectrophoresis after loading of the samples onto a 7.5%SDS-polyacrylamide gel.

Results of this experiment are shown in FIG. 8, which shows the resultsof immunoprecipitations on COS-1 cells transfected with t-BRK-3 in thepresence or absence of BRK-1 or BRK-2. Cells transfected with t-BRK-3alone, crosslinked to [¹²⁵I]-BMP-4, and immunoprecipitated with antibody1380 show no radiolabel in the immunoprecipitate, as expected sincet-BRK-3 does not crossreact with this antibody. Cells transfected withBRK-1, crosslinked, and immunoprecipitated with antibody 1379 show asingle labeled band at 78 kD, consistent with the predicted molecularweight of BRK-1 plus the cross-linked monomer of BMP-4.Immunoprecipitation of cells co-transfected with BRK-1 and t-BRK-3yields the same band seen with BRK-1 alone, plus an additional labeledband at 94 kD, consistent with the predicted molecular weight of t-BRK-3plus the crosslinked BMP-4 monomer. (A less intense band at 120 kD) isalso observed.) The fact that antibodies to BRK-1 precipitate not onlyBRK-1, but t-BRK-3 as well in these cells indicates complex formationbetween BRK-1 and t-BRK-3. Similarly, cells transfected with BRK-2,crosslinked to [¹²⁵I]-BMP-4, and subjected to immunoprecipitation withantibody 1380 show a labeled band at 75 kD, consistent with thepredicted molecular weight of BRK-2 plus the crosslinked monomer ofBMP-4. Immunoprecipitation of cells co-transfected with BRK-2 andt-BRK-3 yields the same band seen with BRK-2 alone, plus a stronglylabeled band at 94 ID, consistent with the predicted molecular weight oft-BRK-3 plus the crosslinked monomer of BMP-4. As expected, this bandco-migrates with the larger labeled band in cells co-transfected withBRK-1 and t-BRK-3. (A less intense band at 120 kD is also observed.)Again, the fact that an antibody to BRK-2 precipitates not only BRK-2but t-BRK-3 as well in these cells strongly indicates that BRK-2 andt-BRK-3 form a complex. Thus, t-BRK-3 forms a complex with two differenttype I BMP receptors, as expected for a type II BMP receptor.

A second immunoprecipitation experiment is carried out to test theligand specificity of the t-BRK-3 receptor complex for BMP-2, BMP-4, andTGF-β₁. A derivative of BMP-2 designated “digit -removed” BMP-2(DR-BMP-2) is also tested; DR-BMP-2 is prepared by mild trypsindigestion of BMP-2 to remove the amino terminus, and shows significantlyreduced nonspecific binding to whole cells (B. B. Koenig, et al., Molec.Cell Biol., 14: 5961-5974 (1994)).

COS-1 cells are co-transfected with the cDNA for BRK-2 and t-BRK-3 asdescribed in Example 5, crosslinked to [¹²⁵I]-BMP-4, and subjected toimmunoprecipitation with antibody 1380 as described in this example,except that an excess of unlabeled ligand (10 nM BMP-4, 10 nM BMP-2, 10nM DR-BMP-2, or 50 nM TGF-β₁) is added to the incubation at the sametime as the [¹²⁵I]-BMP-4. The results are shown in FIG. 9. When nocompeting unlabeled ligands are present, two labeled bands are observed,at 75 kD and 94 kD, consistent with crosslinked BRK-2 and BRK-3respectively, as seen in FIG. 8. In the presence of excess unlabeledBMP-4, BMP-2, or DR-BMP-2, however, these bands are completelyabolished, demonstrating that these ligands compete effectively with[¹²⁵I]-BMP-4 to bind to the complex, and that all these ligands showspecific binding to the BRK-2 and BRK-3 receptor complex. However, thepresence of 50 nM TGF-β₁ has no effect on the labeled bands, indicatingthat TGF-β₁ does not bind to the same site as [¹²⁵I]-BMP-4. This showsthat the BRK-2/t-BRK-3 complex binds specifically to BMP-2 and BMP-4 anddoes not bind TGF-β.

EXAMPLE 9 Isolation of Mouse BRK-3

In order to isolate the full-length mouse homologue of BRK-3, a cDNAlibrary is constructed from NIH3T3 mouse embryonic fibroblasts (ATCC CRL1658). Total RNA (1.26 mg) is isolated from the cells using a Total RNASeparator Kit (Clontech, Palo Alto, Calif.). Messenger RNA (81 μg) isisolated from this total RNA (1 mg) using the mRNA Separator Kit(Clontech, Palo Alto, Calif.). An aliquot of the mRNA (4 μg) is used tomake cDNA library using the SUPER SCRIPT Plasmid System for cDNASynthesis and Plasmid Cloning (Life Technologies, Gaithersburg, Md.)according to the manufacturer's instructions. The resulting librarycontained approximately 4.9×10⁵ primary colonies, and is divided into 98pools, each containing 5000 colonies.

The initial screen of the library is accomplished by Southern blotting.Plasmids are purified from each of the 98 pools, using QIAGEN columns(Qiagen, Chatsworth, Calif.). DNA from each pool (approximately 5 μg) isdigested with Mlu I to release the cDNA insert, then run on a 1% agarosegel. The gel is denatured for 30 min in 0.6 M NaCl, 0.4 N NaOH, thenneutralized 30 min in 1.5 M NaCl, 0.5 M Tris, pH 7.5. The DNA is thentransferred overnight to a HYBOND Nylon membrane (Amersham, ArlingtonHeights, Ill.) using 10× SSC as the transfer buffer (1× SSC 0.15 M NaCl,0.015 M sodium citrate, pH 7.0).

Human t-BRK-3 is cut with EcoRV and Afl III to give a 1.5 kb fragment.The fragment is randomly labeled with alpha[³²P]-dCTP having a specificactivity of 3000 Ci/mmol (NEN Research Products, Boston, Mass.), using aPRIME-IT II Random Primer Labeling Kit (Stratagene, La Jolla, Calif.; akit for random primer labeling of DNA, including Klenow DNA polymerase,primers, and buffers). The labeled probe is allowed to hybridize to theSouthern blot for 18 hr at 42° C. in hybridization buffer (Sigma, St.Louis, Mo.) consisting of 50% deionized formamide, 5× SSPE (1× SSPE=0.14 M NaCl, 8 mM sodium phosphate, 0.08 mM EDTA, pH 7.7), 1×Denhardt's solutions, and 100 μg/ml of denatured salmon testis DNA. Theblot is then washed in 0.25× SSPE, 0.5% sodium dodecyl sulfate (SDS),two times at 42° C. for 15 min each, then two times at 65° C. for 20 mineach. The blot is then exposed to Kodak X-OMAT AR autoradiography filmfor 18 hr at −80° C. Development of the film shows five positive pools,as judged by the presence of a labeled band of approximately 2.5 kb.

For secondary screening, plates are streaked with the E. coli stocksfrom the five positive pools (5000 colonies/plate). A HYBOND nylonmembrane is placed on top of the plate so that the bacterial coloniesare transferred to the filter. The colonies are then allowed to recoverat 37° C. for 2-3 hr. The filter is soaked in 10% SDS for 3 min, thentransferred to 1.5 M NaCl, 0.5 M NaOH for 5 min, neutralized in 1.5 MNaCl, 1.5 M Tris, pH 7.5 for 5 min, and washed in 2× SSC. To removeproteins, the blots are then shaken with 50 μg/ml of proteinase K(Boehringer Mannheim, Indianapolis, Ind.) in 0.1 M Tris, pH 7.6, 10 mMEDTA, 0.15 M NaCl, 0.02% SDS at 55° C. for 1 hr. The human BRK-3fragment (Eco RV-Afl III) is labeled and the blots hybridized, washed,and subjected to autoradiography exactly as described above for theprimary screening.

Colonies which corresponded to labeled spots on the autoradiograph arestreaked on plates for tertiary screening, which is performed exactly asdescribed above for secondary screening. Four positive clones areisolated. One clone, pSPORT1/N89-5, is found to have the largest insertsize, 2.9 kb.

The inserts from the four positive clones are sequenced using the TAQDYE DEOXY Terminator Cycle Sequencing Kit and an Applied BiosystemsModel 373A Automated DNA Sequencer. Comparison of the four sequencesshows that three of the four are identical at the 3′ end, and all fouralign with the coding region of human BRK-3 at the 5′ end. The longestclone, pSPORT1/N89-5, aligns with the human BRK-3 sequence approximately600 pairs from the beginning of the coding region.

To generate more sequence information, the insert from pSPORT1/N89-5 isdigested with EcoRI and Sca I, and the resulting 1.4 kb fragment issubcloned into BLUESCRIPT II SK(−) at the Eco RI and Hinc II sites.pSPORT1/N89-5 is also digested with Eco RI and Eco RV and the resulting2.1 kb insert subcloned into the same vector at the same sites. Finally,the plasmid is digested with Sca I and Not I, and subcloned into thesame vector at the Hinc II and Not I sites. Sequencing of these threeconstructs yields the complete sequence of the insert frompSPORT1/N89-5.

The missing 600 base pairs at the 5′ end of the coding region is clonedusing the 5′ RACE System for Rapid Amplification of cDNA Ends (LifeTechnologies, Gaithersburg, Md.). An antisense primer is designedcorresponding to the known sequence of pSPORT1/N89-5, having thesequence 5′CTG TGT GAA GAT AAG CCA GTC 3′ (SEQ ID NO: 28) (the reversecomplement of nucleotides 968-948 in SEQ ID NO:7). After first strandsynthesis of cDNA from 1 μg of NIH3T3 mRNA, a poly C tail is added tothe newly synthesized cDNA using terminal deoxynucleotidyl transferase,according to the manufacturer's instructions. The primer above is usedto amplify the 5′ end of the BRK-3 cDNA, together with the Anchor Primersupplied with the kit, having the sequence 5′ (CUA)₄ GGC CAC GCG TCG ACTAGT ACG GGI IGG GII GGG IIG 3′ (SEQ ID NO′S: 29, 30, and 31) (whereI=inosine and U=uracil). PCR was performed using the GENE-AMP PCR Kitwith AMPLITAQ DNA Polymerase. An initial melting period at 95° C. for 5min was followed by 35 cycles of the following program: melting at 95°C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for2 min. After the last cycle, the reaction was held at 72° C. for 5 minto complete extension. To reduce background from nonspecific primerbinding, a second round of PCR is performed using the nested primer5′CAA GAG CTT ACC CAA TCA CTT G 3′ (SEQ ID NO: 32), again derived fromthe known sequence of the insert from pSPORT1/N89-5 (the reversecomplement of nucleotides 921-900 in SEQ ID NO: 7), together with same5′ anchor primer used in the first round of PCR.

The amplified products of the second PCR reaction in the size range of600-1000 bp are digested with Ecl XI and Sal I and subcloned intoBLUESCRIPT II SK(−) at the Ecl XI and Sal I sites. The inserts are thensequenced, yielding an additional 600 bp of sequence which align withthe coding region of human t-BRK-3. Three separate clones, designatedR6-8B2, R6-11-1, and R6-11-2, are sequenced with identical results.

In order to assemble a full length clone of mouse BRK-3, a Sal I site isfirst placed at the 5′ end of clone R6-11-1 as follows. A primer issynthesized which contains a Sal I site followed by nucleotides 1-20 ofthe sequence of R6-11-1; the sequence of the primer is 5′CAC ACG CGT CGACCA TGA CTT CCT CGC TGC ATC G 3′ (SEQ ID NO: 33). This is used togetherwith the M13 reverse primer, 5′ AAC AGC TAT GAC CAT G 3′ (SEQ ID NO:34), in order to amplify a DNA fragment using plasmid DNA from cloneR6-11-1 as the template. PCR was performed using the GENE-AMP PCR Kitwith AMPLITAQ DNA Polymerase. An initial melting period at 95° C. for 5min was followed by 35 cycles of the following program: melting at 95°C. for 1 min, annealing at 55° C. for 1 min, and extension at 72° C. for2 min. After the last cycle, the reaction was held at 72° C. for 5 minto complete extension. The fragment amplified from R6-11-1, togetherwith the insert from pSPORT1/N89-5 (230 ng), is then subcloned in toBLUESCRIPT II SK(−) as follows. The amplified fragment from R6-11-1 isdigested with Sal I and Ecl XI. The insert from pSPORT1/N89-5 isdigested with Ecl XI and Pst I. The vector BLUESCRIPT II SK(−) isdigested with Sal I and Pst I. The three fragments are combined in athree-way ligation using T4 DNA ligase (3 hr, 25° C.) and used totransform electrocompetent E. coli, strain DH5-a, using a BIO-RAD GenePULSER (BIO-RAD, Hercules, Calif.) according to the manufacturer'sinstructions. A positive colony is selected and is designatedpBLUESCRIPT-mBRK3. Sequencing of the 5′ portion of the insert that wasamplified by PCR shows a sequence identical to that of clone R6-11-1,indicating that no mutations are introduced during the amplification.

For mammalian expression, m-BRK-3 is subcloned into the mammalianexpression vector pJT6. This vector is a derivative of pJT3, describedin example 4 above, in which the Not I site at the 5′ end of themultiple cloning site has been deleted, and a spacer inserted betweenthe Pst I and BamHI restriction sites in the multiple cloning site. Toaccomplish the subcloning, m-BRK-3 is excised from pBLUESCRIPT-mBRK3using Not I and Sal I, then subcloned into pJT6 at the Not I and Sal Isites to generate pJT6-mBRK3.

However, resequencing of the 3′ end of pJT6-mBRK3 and the original cDNAin pSPORT1/N89-5 results in an altered reading frame at the 3′ end, andshows that the stop codon is actually located 3′ to the Pst I site.Thus, pJT6-mBRK3 does not contain a stop codon. Accordingly, two newconstructs are prepared as follows.

First, pJT6-mBRK3 is digested with SpeI (site at position 2306 in SEQ IDNO: 7) and Not I (in the multiple cloning site of pJT6), removing the 3′end of the insert. The longest clone isolated during the screening ofthe NIH-3T3 library, pSPORT1/N89-5, is also digested with Spe I and NotI. The 1.2 kb fragment liberated from pSPORT1/N89-5 is subcloned intothe Spe I/Not I digested pJT6-mBRK3, regenerating both sites. Thisconstruct is designated pJT6-mBRK-3L, and contains the entire 3′ end ofthe pSPORT1/N89-5 clone. A map of the construct is shown in FIG. 10.

The 3′ end of the clone contains 403 nucleotides in the untranslatedregion 3′ to the stop codon. This region is very A-T rich, which mightpossibly lead to decreased expression levels. To remove this region, asecond construct is prepared. The pSPORT1/N89-5 plasmid is digested withHind III (site at nucleotide 3168 in SEQ ID NO: 7, 21 bases 3′ to thestop codon). The linearized plasmid is treated with Klenow fragment ofDNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) to fill inoverhangs, then cut with Spe I to liberate an 863 bp fragment at the 3′end of the insert. At the same time, pJT6-mBRK3 is digested with Not I.The linearized plasmid is treated with Klenow fragment, then cut withSpe I, releasing the 3′ end of the insert. The Not I/Spe I digestedpJT6-mBRK3 is then ligated to the fragment liberated from pSPORT1/N89-5by Hind III/Spe I. This regenerates the Spe I site; the Hind III and NotI sites are destroyed. The resulting construct is designatedpJT6-mBRK3S, and is shown in FIG. 1.

The construct pJT6-mBRK-3S is also constructed directly from the partialcDNA clone of m-BRK-3, pSPORT1/N89-5, and the construct containing the5′ end of the cDNA, clone R6-11-1. This is accomplished by digestion ofclone R6-11-1 with Sal I and Ecl XI, digestion of pSPORT1/N89-5 with EclXI and Hind III, and digestion of BLUESCRIPT II SK (−) with Sal I andHind III. These fragments are then subjected to a three-way ligation togenerate the full length m-BRK-3 cDNA in the BLUESCRIPT II vector. Thefull length cDNA is then excised from this construct using Sal I and NotI, then subcloned into the Sal I and Not I sites of the pJT6 vector. Theresulting plasmid has exactly the same cDNA for BRK-3 as does pJT6-mBRK3S described in the above example. However, it carries additional vectorsequence at the 3′ end of the cDNA, comprising the region between theHind III and Not I sites in the multiple cloning site of BLUESCRIPT IISK(−).

EXAMPLE 10 Sequence Analysis of Mouse BRK-3

The DNA sequence of the full length mouse BRK-3 insert from pJT6-mBRK3Lis shown in SEQ ID NO: 7, and the deduced protein sequence is shown inSEQ ID NO: 8. The deduced amino acid sequence of mouse BRK-3 is searchedagainst all translated protein sequences in GenBank release 84.0, datedAug. 15, 1994, using a standard Needleman-Wunsch algorithm (S. B.Needleman and C. D. Wunsch, J. Mol. Biol., 48: 443-453 (1970)). It isfound to be a unique sequence. It encodes a protein of 1038 amino acids.Comparing mouse BRK-3 with the truncated human receptor over the regionencoded by t-BRK-3 (amino acids 1-582 in SEQ ID NO:4; amino acids 1-582in SEQ ID NO: 8), the two receptors are 98% identical in sequence. Liket-BRK-3, m-BRK-3 contains a predicted transmembrane region encompassingamino acids 151-172. As with t-BRK-3, the intracellular domain containsall of the consensus sequences that characterize a protein kinase domainwith predicted specificity for serine/threonine residues (S. K. Hanks,A. M. Quinn, and T. Hunter, Science, 241: 42-52 (1988)). The kinasedomain is followed by an extremely long carboxy terminus (534 aminoacids). Indeed, due to the presence of this carboxy terminus, theintracellular domain in BRK-3 (866 amino acids) is much larger than thatof any other receptor in the TGF-β receptor family. It is nearly twiceas long as the intracellular domain of DAF-4 (490 amino acids), whichhas the longest intracellular domain known in the TGF-β family until thepresent invention.

EXAMPLE 11 Demonstration of [¹²⁵I]-BMP-4 Binding to m-BRK-3

In order to demonstrate that [¹²⁵I]-BMP-4 binds specifically to m-BRK-3,COS-1 cells are transfected as described in Example 5 using theconstructs pJT6-mBRK-3S and pJT6-mBRK-3L. In addition, the cells arealso co-transfected with cDNA for the type I receptor BRK-2, using theconstruct pJT3-BRK-2, to determine whether the presence of a type I BMPreceptor affects binding of [¹²⁵I]-BMP-4. Whole cell binding with[¹²⁵I]-BMP-4 is carried out as described in Example 7.

The results are shown in FIG. 12, which shows specific binding of[¹²⁵I]-BMP-4 normalized to cell number. When cells are transfected withmouse BRK-3 alone, using either of the two constructs tested, specificbinding of [¹²⁵I]-BMP-4 is increased to 4-7 times the level seen withmock transfected cells. Transfection of BRK-2 alone shows increasedbinding at a similar level to that seen with mouse BRK-3 alone. Whencells are co-transfected with BRK-2 as well as mouse BRK-3, the bindingis further increased to 9-11 times that of mock-transfected cells,consistent with the results obtained with BRK-2 in combination witht-BRK-3 (FIG. 6 in Example 7 above).

As an additional demonstration that m-BRK-3 binds to[¹²⁵I]-BMP-4, acrosslinking experiment is carried out. COS-1 cells are transfected withthe cDNA for m-BRK-3, using the construct pJT6-mBRK-3S, and/or withcDNAs for BRK-1 (using pJT4-J159F) or BRK-2 (using pJT3-BRK-2) asdescribed in Example 5. The transfected cells are incubated with[¹²⁵I]-BMP-4 and crosslinked as described in Example 7, except thatdisuccinimidyl suberate (DSS) is used as the crosslinking agent ratherthan disuccinimidyl glutarate. The results of such an experiment areshown in FIG. 13. Cells transfected with m-BRK-3 alone show nocrosslinked band, consistent with the results obtained with t-BRK-3(FIG. 7). Cells transfected with the cDNA for BRK-1 alone show a singlespecies migrating at an apparent molecular weight of 81 kD, consistentwith the predicted molecular weight of BRK-1 plus the crosslinked BMP-4monomer. Cells transfected with the cDNAs for BRK-1 and m-BRK-3 showthree labeled bands, one of which is consistent with the band seen withBRK-1 alone (81 kD). The other bands migrate with an apparent molecularweight of 159 kD and 128 kD. The larger of these is consistent with thepredicted molecular weight of m-BRK-3 plus the crosslinked BMP-4monomer. Note that the intensity of the crosslinked band identified withBRK-1 is considerably increased, compared to that seen with BRK-1 alone.

Similarly, transfection of cells with the cDNA for BRK-2 alone yields acrosslinked band migrating at an apparent molecular weight of 78 kD,consistent with the predicted molecular weight of BRK-2 plus thecrosslinked BMP-4 monomer. In cells transfected with the cDNAs for BRK-2and mBRK3, the 78 kD species identified with BRK-2 is observed, as wellas crosslinked bands at 159 kD and 128 kD, comigrating with the highermolecular weight bands seen in cells transfected with the cDNAs forBRK-1 and m-BRK-3. As with BRK-1, the intensity of crosslinking to theband identified with BRK-2 is considerably increased compared to thatseen with BRK-2 alone. Finally, no labeled bands are observed in cellstransfected with vector alone.

An immunoprecipitation experiment is carried out to demonstrate theability of m-BRK-3 to form a complex with type I BMP receptors. COS-1cells are transfected with the cDNA for m-BRK-3, using the constructpJT6-mBRK-3S, and/or with cDNAs for BRK-1 (using pJT4-J159F) or BRK-2(using pJT3-BRK-2) as described in Example 5. The transfected cells areincubated with [¹²⁵I]-BMP-4, crosslinked, and subjected toimmunoprecipitation with antibodies to the appropriate type I receptoror preimmune serum as described in example 8, except that DSS is used asthe crosslinking agent rather than disuccinimidyl glutarate. The resultsof this experiment are shown in FIG. 14. In cells transfected with cDNAfor BRK-1 alone, a single band is precipitated by antibodies to BRK-1,migrating at an apparent molecular weight of 81 kD. In cells transfectedwith cDNAs for BRK-1 and m-BRK-3, antibodies to BRK-1 precipitate the 81kD band, which is now increased in intensity. In addition, however, aband migrating at an apparent molecular weight of 159 kD is observed,consistent with the predicted molecular weight of m-BRK-3 pluscrosslinked BMP-4 monomer. Similarly, in cells transfected with cDNA forBRK-2 alone, antibodies to BRK-2 precipitate a labeled species migratingat an apparent molecular weight of 78 kD. In cells transfected withcDNAs for BRK-2 and m-BRK-3 and precipitated with antibodies to BRK-2,the 78 kD band identified with BRK-2 is again observed, at increasedintensity. In addition, a labeled species is seen at 159 kD, consistentwith m-BRK-3 and comigrating with the higher molecular weight band seenin cells transfected with cDNAs for BRK-1 and m-BRK-3. In cellstransfected with cDNAs for BRK-2 and m-BRK-3, an additional labeled bandis observed at 94 kD. As a control, cells are transfected with the cDNAsfor BRK-1 and m-BRK-3, or BRK-2 and m-BRK-3, then subjected toimmunoprecipitation with preimmune sera (lanes far left and far right);no labeled bands are observed.

This experiment shows that when m-BRK-3 is co-expressed with the type IBMP receptors BRK-1 or BRK-2, antibodies which precipitate the type Ireceptor also precipitate m-BRK-3. Thus, m-BRK-3 can form a complex witheither of these mammalian type I BMP receptors, as expected for amammalian type II BMP receptor. This is consistent with results obtainedwith t-BRK-3 described in Example 8 above.

EXAMPLE 12 Isolation of Full Length Human BRK-3 cDNA

Since clone HSK723, described in Example 2, does not contain an in-framestop codon, it is desired to obtain additional sequence 3′ to the end ofthis cDNA. Accordingly, the human foreskin fibroblast library preparedin Example 1 is rescreened with the HSK7-2 PCR fragment, using labelingand screening conditions exactly as described in Example 2. This resultsin isolation of a longer clone, designated pHSK1030, which containsadditional human BRK-3 sequence (total of 3355 base pairs) subcloned inBLUESCRIPT SK(−). Sequencing of the insert from pHSK1030 discloses acoding region of 982 amino acids, but the insert still does not containan in-frame stop codon.

The remainder of the coding region is cloned by PCR as follows. Twoforward primers are derived from the plus strand of clone pHSK1030. Thesequences of these primers are as follows: primer RPK3-1,5′CCTGTCACATAATAGGCGTGTGCC-3′ (SEQ ID NO: 35) (identical to nucleotides1998-2021 in SEQ ID NO:1); primer RPK3-2, 5′ CGCGGATCCATCATACTGACAGCATCG3′ (SEQ ID NO: 36) (which incorporates a BamHI site followed bynucleotides 2078-2095 in SEQ ID NO:1). Two additional primers arederived from the minus strand of λgt10. These primers are: G10F1, 5′GCTGGGTAGTCCCCACCTTT 3′ (SEQ ID NO: 37)and G10F2, 5′ GAGCAAGTTCAGCCTGGT3′ (SEQ ID NO: 38).

The human fibroblast cDNA library prepared in Example 1 is used as thetemplate for PCR. The library (0.3 μg) is incubated with the RPK3-1 andG10F1 primers (1 μM each), Tth polymerase (1.2 units), all fourdeoxynucleotides (200 μM each), buffer for the Tth polymerase, and waterin a total of 50 μl. Conditions for the PCR cycle are as follows:initial melting at 94° C. for 2 min, followed by 20 cycles of melting,94° C. for 1.5 min; annealing, 52° C. for 2 min; and extension, 72° C.for 3 min. After cycle 20, the sample is held at 72° C. for anadditional 8 min to insure complete extension.

To increase specificity and reduce background, a second round of nestedPCR is carried out. The incubation mixture is the same as described inthis example for the first round, except that (1) an aliquot of thefirst PCR reaction (0.5 μl) is used as the template; and (2) RPK3-2 andG10F2 primers are used, instead of RPK3-1 and G10F1. Conditions for thePCR run are identical to those described in this example for the firstround of PCR.

The second round of PCR results in the amplification of a 1.6 kbfragment, which is isolated from an agarose gel by QIAEX. This fragmentis digested with EcoRI and BamHI, and subcloned into BLUESCRIPT SK(−) atthe EcoRI and Bam HI sites. The resulting construct, pHSK723-3U, issequenced and found to encode the remaining coding region of BRK-3 withan in-frame stop codon.

In order to assemble the full length human BRK-3, the inserts frompHSK1030 and pHSK723-3U are joined at a unique Stu I site (located atnucleotide 3219 in SEQ ID NO: 1) in the vector BLUESCRIPT II SK(−). Thisyields the complete construct pHSK1040, which contains the completecoding sequence of human BRK-3. The pHSK 1040 is shown in FIG. 15. TheDNA sequence of human BRK-3 is shown in SEQ ID NO:1, and the deducedamino acid sequence for human BRK-3 is shown in SEQ ID NO: 2.

The amino acid sequence of human BRK-3 (SEQ ID NO:2) is compared to theamino acid sequence for m-BRK-3 (SEQ ID NO:8) and found to be 96.7%identical.

EXAMPLE 13 Use of the BRK-3 in a Ligand Binding Assay For theIdentification of BMP Receptor Agonists and Antagonists

Identification of ligands that interact with BRK-3 can be achievedthrough the use of assays that are designed to measure the interactionof ligands with BRK-3. An example of a receptor binding assay that isadapted to handle large numbers of samples is carried out as follows.

COS-1 cells are transfected with the cDNA for m-BRK-3 using theconstruct pJT6-mBRK-3L as described in example 11 above, except thatcells are grown in a 12 well culture dish. At 48-68 hr aftertransfection, the cells are washed once with 1.0 ml binding buffer (50mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCL, 5 mM MgSO₄, 1.2 mM CaCl₂, 2mg/ml BSA), then equilibrated in the same buffer at 4° C. for 60 min.with gentle shaking. After equilibration, the buffer is aspirated, andto each well is added 500 μl of 4° C. binding buffer containing[¹²⁵I]BMP-4 tracer (100-400 pM) in the presence or absence of varyingconcentrations of unlabeled test compounds (i.e., putative ligands), fora period of 4 hours at 4° C. with gentle shaking. For determination ofnonspecific binding and complete displacement from the BMP receptorcomplex, BMP-2 is added at a final concentration of 10 nM. To preventdegradation of ligand, a protease inhibitor cocktail is also added, togive a final concentration of 10 μg/ml leupeptin, 10 μg/ml antipain, 50μg/ml aprotinin, 100 μg/ml benzamidine, 100 μg/ml soybean trypsininhibitor, 10 μg/ml bestatin, 10 μg/ml pepstatin, and 300 μMphenylmethylsulfonyl fluoride (PMSF). At the end of the incubationperiod, the buffer is aspirated, and the cells are rinsed 4 times with 1ml washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCl, 5 mMMgSO₄, 1.2 mM CaCl₂, 0.5 mg/mil BSA). After the final wash is aspirated,200 μl of solubilization buffer (10 mM Tris Cl, pH 7.4, 1 mM EDTA, 1%(v/v) Triton X-100) is added to each well and incubated at roomtemperature for 15-30 min. The solubilized cells are then transferred tofresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter(Packard Instruments, Meriden, Conn.).

Test compounds which interact with the m-BRK-3 receptor are observed tocompete with binding to the receptor with the [¹²⁵I]BMP-4 tracer in thecells expressing m-BRK-3, such that less [¹²⁵I]BMP-4 tracer is bound inthe presence of the test compound in comparison to the binding observedwhen the tracer is incubated in the absence of the novel compound. Adecrease in binding of the [¹²⁵I]BMP-4 tracer by ≧30% at the highestconcentration of the test compound that is studied demonstrates that thetest compound binds to m-BRK-3.

Similar results are obtained when other, related BRK-3 protein receptorkinases of the present invention are used according to the method ofthis example.

EXAMPLE 14 Use of m-BRK-3 and BRK-2 in a Ligand Binding Assay For theIdentification of BMP Receptor Agonists and Antagonists

Identification of ligands that interact with BRK-3 complexed to a type IBMP receptor can be achieved through the use of assays that are designedto measure the interaction of the ligands with this BMP receptorcomplex. A receptor binding assay that uses the m-BRK-3/BRK-2 complexand is adapted to handle large numbers of samples is carried out asfollows.

COS-1 cells are transfected with the cDNAs for m-BRK-3, using theconstruct pJT6-mBRK-3L, and BRK-2, using the construct pJT3-BRK-2, asdescribed in example 11 above, except that the cells are grown in a 12well culture dish. The DNA mixture used to transfect the cells contains2 μg/ml of pJT3-BRK-2 and 4 μg/ml of pJT6-mBRK-3L. At 48-68 hours aftertransfection, the cells are washed once with 1 ml binding buffer (50 mMHEPES, pH 7.4, 128 mM NaCl, 5 mM KCL, 5 mM MgSO₄, 1.2 mM CaCl₂, 2 mg/mlBSA), then equilibrated in the same buffer at 4° C. for 60 min withgentle shaking. After equilibration, the buffer is aspirated, and toeach well is added 500 μl of 4° C. binding buffer containing [¹²⁵I]BMP-4tracer (100-400 pM) in the presence or absence of varying concentrationsof test compounds (i.e., putative ligands), for a period of 4 hours at4° C. with gentle shaking. For determination of nonspecific binding andcomplete displacement from the BMP receptor complex, BMP-2 is added at afinal concentration of 10 nM. To prevent degradation of ligand, aprotease inhibitor cocktail is also added, to give a final concentrationof 10 μg/ml leupeptin, 10 μg/ml antipain, 50 μg/ml aprotinin, 100 μg/mlbenzamidine, 100 μg/ml soybean trypsin inhibitor, 10 μg/ml bestatin, 10μg/ml pepstatin, and 300 μM phenylmethylsulfonyl fluoride (PMSF). At theend of the incubation period, the buffer is aspirated, and the cells arerinsed 4 times with 1 ml washing buffer (50 mM HEPES, pH 7.4, 128 mMNaCl, 5 mM KCl, 5 mM MgSO₄, 1.2 mM CaCl₂, 0.5 mg/ml BSA). After thefinal wash is aspirated, 200 μl of solubilization buffer (10 mM Tris Cl,pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100) is added to each well andincubated at room temperature for 15-30 min. The solubilized cells arethen transferred to fresh tubes and counted in a Packard Model 5005COBRA Gamma Counter (Packard Instruments, Meriden, Conn.).

Test compounds which interact with the m-BRK-3/BRK-2 receptor complexare observed to compete for binding to the receptor complex with the[¹²⁵I]BMP-4 tracer, such that less [¹²⁵I]BMP-4 tracer is bound in thepresence of the test compound in comparison to the binding observed whenthe tracer is incubated in the absence of the novel compound. A decreasein binding of the [¹²⁵I]BMP-4 tracer by >30% at the highestconcentration of the test compound that is studied demonstrates that thetest compound binds to the m-BRK-3/BRK-2 receptor complex.

Similar results are obtained when the other BRK-3 protein receptorkinases of the present invention, or homologues thereof, are used incombination with BRK-2 or other BMP type I receptors.

EXAMPLE 15 Use of m-BRK-3 and BRK-2 in a Signaling Assay For theIdentification of BMP Receptor Agonists and Antagonists

Identification of ligands that signal upon interaction with BRK-3complexed to a type I receptor can be achieved through the use of assaysthat are designed to measure the activation of the receptor proteinkinase domain after binding of the ligand to the receptor complex. An invivo phosphorylation assay that measures changes in phosphorylation ofproteins that are immunoprecipitated by antibodies to the BRK-2 Type Ireceptor in cells that express the mBRK-3/BRK-2 complex is carried outas follows.

COS-1 cells are transfected with the cDNAs for m-BRK-3, using theconstruct pJT6-mBRK-3S, and BRK-2, using the construct pJT3-BRK-2, asdescribed in Example 11 above, except that cells are grown in a T175flask (Falcon). The DNA mixture used to transfect the cells contains 2μg/ml of pJT3-BRK-2 and 4 μg/ml of pJT6-mBRK-3S. 24 hours aftertransfection, the cells are plated into 100 millimeter tissue culturedishes, allowed to attach to the plates for at least 5 hours, and thenthe media is changed to DMEM (Life Technologies, Inc., Gaithersburg,Md.) containing 1% fetal bovine serum (HyClone, Logan, Utah), and 2 mML-glutamine, 0.1 mM MEM nonessential amino acids solution; and grown inthis low serum media for an additional 12-18 hours. These serum-starvedcells are washed three times with ten milliliters per dish ofphosphate-free Dulbecco's modified Eagle's medium (DMEM, LifeTechnologies, Inc., Gaithersburg, Md.) supplemented with 2 mML-glutamine, 0.1 mM MEM nonessential amino acids solution, and 25 mMHEPES buffer, pH 7.4. Three milliliters per dish of supplementedphosphate-free DMEM and 0.8-1.0 milliCuries per dish of [³²P]orthophosphoric acid (DuPont New England Nuclear, Wilmington, Del.) arethen added to the cells, which are incubated at 37° C. in 95% air/5% CO₂for 3 hours. Following incubation, the cells are treated withappropriate concentrations of ligand stimulators such as BMP-4 for 5minutes at 37° C. Treated cells are washed three times with tenmilliliters per dish of 4° C. 50 mM Tris-buffered saline solution(Sigma, St. Louis, Mo.) and lysed for ten minutes at 4° C. in onemilliliter per dish of P-RIP buffer (20 mM Tris pH 8.0, 100 mM sodiumchloride, 1 mM disodium ethylenediaminetetraacetic acid, 0.5% NonidetP-40, 0.5% sodium deoxycholate, 10 mM sodium iodide, 1% bovine serumalbumin, 50 mM sodium fluoride, 30 mM tetrasodium pyrophosphate, 250 mMsodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride (allreagents from Sigma, St. Louis, Mo.)). The lysates are clarified bycentrifugation at 10,000×g for ten minutes and sodium dodecyl sulfate(Sigma, St. Louis, Mo.) is added to 0.1% final concentration from a 10%stock solution (the resulting buffer is called P-RIPS: P-RIPsupplemented with 0.1% sodium dodecyl sulfate). 100 μl of PANSORBIN (a10% solution of S. aureus cells; Pansorbin: Calbiochem, La Jolla,Calif.) is added and tubes are incubated for 30 minutes on ice. ThePANSORBIN is removed by centrifugation at 10,000×g for three minutes andthe supernatants are transferred to new tubes containing a 1:100dilution of rabbit anti-BRK-2 polyclonal antisera obtained as describedin Example 8 above, except that the BRK-2 antibody that is used for thisassay is generated against the peptide ARPRYSIGLEQDETYIPPC, (SEQ ID NO:39), which is based on the amino acid sequence of BRK-2 in theintracellular juxtamembrane region, comprising amino acids 155-172 inSEQ ID NO:14, with the addition of a cysteine at the C terminus topermit conjugation of the peptide, as described in Example 8, above.Following an overnight incubation at 4° C., 50 microliters of Pansorbinis added and incubated for an additional 30 minutes at 4° C. ThePansorbin-bound complexes are pelleted by centrifugation at 10,000×g forthree minutes and the pellets are washed three times with P-RIPS bufferand once with P-TNEN buffer (20 mM Tris pH 8.0, 100 mM sodium chloride,1 mM disodium ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, 50 mMsodium fluoride, 30 mM tetrasodium pyrophosphate, 250 mM sodiumorthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The pellets arethen resuspended in 20 microliters per tube of SDS-PAGE sample buffer (5Prime-3 Prime, Boulder, CO: 50 mM Tris pH 6.8, 2% sodium dodecylsulfate, 0.1 M dithiothreitol, 0.1% bromophenol blue, 10% glycerol),heated at 95° C. for five minutes, and pelleted by centrifugation at10,000×g for three minutes. The supernatants are electrophoresed througha 7.5%, 12.5%, or 15% SDS-polyacrylamide gel (Integrated SeparationSystems, Natick, Mass.) at a current of 35 milliamps per gel in anelectrophoretic running buffer consisting of 25 mM Tris pH 8.5, 192 mMglycine, and 0.1% sodium dodecyl sulfate (Integrated Separation Systems,Natick, Mass.). The gels are fixed for 15 minutes in a 40% methanol/10%acetic acid solution, dried, and either exposed for autoradiography at−80° C. or subjected to Phosphorimager analysis (Molecular Dynamics,Sunnyvale, Calif.).

Test compounds which are agonists of the BRK-2/BRK-3 receptor complexwill cause an increase in phosphorylation of the proteinsimmunoprecipitated by antibodies to the BRK-2 receptor, as judged by anincreased labeling of the proteins with [³²P]. In order to test forantagonist activity, test compounds are added in the presence of a fixedconcentration of BMP-4 or another BMP receptor agonist. Test compoundswhich are antagonists of the BRK-2/BRK-3 complex will cause a decreasein the labeling of the proteins present in the BRK-2 immunoprecipitatein comparison to that observed after stimulation of the cells with onlythe fixed concentration of BMP-4, or another BMP receptor agonist.

Deposit of BRK-3, t-BRK-3 and m-BRK-3

E. coli transformed with pJT4-J159F (SEQ ID NO:11 subcloned intoexpression vector pJT4) was deposited with the ATCC on Oct. 7, 1993, andassigned ATCC Designation No. 69457.

E. coli transformed with pJT4-hBRK3T (SEQ ID NO:3 subcloned intoexpression vector pJT4) was deposited with the ATCC on Aug. 16, 1994 andassigned ATCC designation No. 69676.

E. coli transformed with pJT6-mBRK-3S (SEQ ID NO: 7 subcloned intoexpression vector pJT6) was deposited with the ATCC on Sep. 28, 1994 andassigned ATCC designation No. 69694.

E. coli transformed with pJT6-mBRK-3L (SEQ ID NO:7 subcloned intoexpression vector pJT6) was deposited with the ATCC on Sep. 28, 1994 andassigned ATCC designation No. 69695.

E. coli transformed with pHSK1040 (SEQ ID NO:1 subcloned into BLUESCRIPTII SK(−) was deposited with the ATCC on Oct. 12, 1994, and assigned ATCCdesignation No. 69703.

As is recognized in the art, there are occasionally errors in DNA andamino acid sequencing methods. As a result, the sequences encoded in thedeposited material are

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to one skilled in the art and are tobe included in the spirit and purview of this application and scope ofthe appended claims.

1. A host cell co-transfected with an expression vector comprising a DNAsequence that codes for a BMP receptor kinase protein BRK-3, wherein theDNA sequence that codes for the BMP receptor kinase protein BRK-3 isselected from the group consisting of SEQ ID NO:1; SEQ ID NO:3; SEQ IDNO:5; SEQ ID NO:7; SEQ ID NO:9, and an expression vector comprising aDNA sequence that codes for a BMP type I receptor kinase protein,wherein the DNA sequence that codes for the BMP type I receptor kinaseprotein is selected from the group consisting of SEQ ID NO:11 and SEQ IDNO:13.
 2. The host cell of claim 1, wherein the DNA sequence that codesfor the BMP receptor kinase protein BRK-3 is SEQ ID NO:3.
 3. The hostcell of claim 2, wherein the DNA sequence that codes for the BMP type Ireceptor kinase protein is SEQ ID NO:13.
 4. The host cell of claim 1,wherein the DNA sequence that codes for the BMP receptor kinase proteinBRK-3 is SEQ ID NO:7.
 5. The host cell of claim 4, wherein the DNAsequence that codes for the BMP type I receptor kinase protein is SEQ IDNO:13.